Tumor vaccination involving a humoral immune response against self-proteins

ABSTRACT

The present invention relates to tumor immunotherapy, in particular to tumor vaccination, using chimeric proteins comprising all or a portion of a hepatitis B virus core antigen protein and an amino acid sequence comprising an epitope derived from the extracellular portion of a tumor-associated antigen. In particular, the present invention provides virus-like particles comprising said chimeric proteins, which are useful for eliciting a humoral immune response in a subject against the tumor-associated antigen, in particular against cells carrying said tumor-associated antigen on their surface, wherein the tumor-associated antigen is a self-protein in said subject.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/457,897, filed on Aug. 12, 2014, which is a continuation of U.S.application Ser. No. 13/634,696, filed on Nov. 6, 2012, now U.S. Pat.No. 8,840,902, which is a U.S. National Stage Application ofInternational Application Serial Number PCT/EP2011/001168, filed on Mar.9, 2011, which claims priority to European Application Serial No.10016216.3, filed on Dec. 30, 2010 and European Application Serial No.10002775.4, filed on Mar. 16, 2010. The entire contents of theabove-referenced applications are incorporated here by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention is in the field of tumor immunotherapy. Inparticular, the present invention provides means for effectivevaccination of a subject against tumor-associated antigens which areself-proteins in said subject. The present invention provides a proteincomprising a hepatitis B virus core antigen protein or a portion thereofand an amino acid sequence comprising an epitope of a tumor-associatedantigen. Furthermore, the present invention provides virus-likeparticles comprising said protein and an immunogenic compositioncomprising said protein or said particles, in particular, for use inprophylactic and/or therapeutic applications, for example, for cancervaccination and/or therapy. The present invention also provides methodsfor breaking self-tolerance against the above tumor-associated antigensand for treating and/or preventing a tumorigenic disease in a subject.

BACKGROUND OF THE INVENTION

Recombinant vaccines are of particular importance in human andveterinary medicine for prophylaxis and therapy of infectious andcancerous diseases. It is the aim of an immunization with a recombinantvaccine to induce a specific immune reaction against a defined antigen,which is effective in prevention or therapy of defined diseases. Knownrecombinant vaccines are based on recombinant proteins, syntheticpeptide fragments, recombinant viruses, or nucleic acids.

Most of the recombinant vaccines can be divided into two categories: a)vaccines inducing a humoral B cell-mediated immune response which resultin specific antibody production, and b) vaccines inducing cellularT-cell mediated immune responses, in particular cytotoxic T-lymphocytes.

Induction of antibodies by preventive vaccination against infectiousdiseases (e.g., vaccinations against children's diseases) is one of themost effective medical interventions and has been applied successfullyfor many years. Recently, it also has been shown that the therapeuticpassive administration of monoclonal antibodies (mAb) directed againstself-proteins represents an effective therapy method of acute andchronic diseases such as cancers or rheumatoid arthritis. Examples formAb targeted structures are the soluble protein tumor necrosis factoralpha (TNF-α) for rheumatoid arthritis, Crohn's disease and psoriasis(mAb preparation: Infliximab and Adalimumab), as well as the cellsurface proteins CD20 for non-Hodgkin lymphoma (mAb preparation: e.g.,Rituximab) and HER2/neu receptor (mAb preparation: Trastuzumab[Herceptin]) for breast cancer.

The generation of monoclonal immunotherapeutically effective antibodies(using hybridoma or phage display techniques and subsequentchimerization and humanization, respectively), however, is timeconsuming and cost intensive which has prevented a broad clinicalapplication so far. Thus, there is an urgent need to provide apossibility for active vaccination against self-molecules instead of thepassive administration of monoclonal antibodies. In contrast to passiveimmunization, during active vaccination the patient's own immune systemis induced to produce antibodies. The induced individualized immuneresponse thus circumvents problems of the monoclonal antibody therapysuch as intolerance or non-responsiveness to the therapy.

The active induction of a humoral immune response against self-proteins,however, requires that the immunological self-tolerance is broken.Self-proteins or peptides thereof are only very weekly immunogenic dueto the immunological tolerance against self-proteins. Existingimmunization strategies based on recombinant proteins or syntheticpeptide fragments for induction of antibody responses againstself-proteins are thus based on concomitant administration of theantigen in combination with immunostimulatory adjuvants. Many potentlyeffective adjuvants, however, exhibit the disadvantage of undesirableside effects such as toxicity, inflammation reactions, or unwantedsystemic T-cell response, and thus, their use should be avoided foractive vaccination strategies.

There are certain requirements for an active immunotherapeuticallyeffective vaccination such as breaking self-tolerance againstself-proteins, avoidance of adjuvants, antibody specificity againstproteins in their native conformation, and induction of antibodies withimmune effector functions.

Another essential factor for a successful active vaccination in thecontext of an antibody-mediated cancer immunotherapy is the selection ofan appropriate tumor target structure.

Basic requirements for the target structure are tumor-specificity andcell surface localization. This allows for selective binding of theinduced antibodies to the tumor cells and allows for directed exertionof effector functions of the antibody against these cells. Particularlyinteresting tumor-associated antigens are the so-called cell typespecific differentiation antigens. Their expression is limited to cellsof a particular specificity and developmental stage in normal tissues.However, in many cancerous diseases, these antigens are expressed in thetumorigenic tissue.

There is an urgent need for the development of means which allow forself-tolerance breaking active immunization without the need ofadministering adjuvants. In particular, there is a need for thedevelopment of means that allow for the generation of antibodies witheffector functions, such as antibody-dependent cellular cytotoxicity(ADCC), complement-dependent cytotoxicity (CDC), induction of apoptosis,and inhibition of proliferation, in vivo, wherein said antibodies aredirected against a self-protein, such as a tumor-associated antigen.

The present invention relates to the development of vaccines for activevaccination which are able to induce antibodies, in particularautoantibodies, in an organism which bind to self cell membrane surfaceantigens in their native conformation and subsequently exerttherapeutically effective effector functions on cells carrying said cellmembrane surface antigens.

The development of cancer immunotherapeutic vaccines is exemplarilydescribed for the target structures claudin 18.2 (CLDN18.2), claudin 6(CLDN6), and PLAC1, respectively. The generated vaccines are capable ofinducing an effective humoral immune response which breaks the presentimmunological self-tolerance, without concomitant administration ofadjuvants. The induced antibodies are further able to recognize theproteins in their native conformation and exert therapeutically relevanteffector functions such as ADCC and/or CDC.

SUMMARY OF THE INVENTION

In a one aspect, the present invention provides a protein comprising allor a portion of the amino acid sequence of a hepatitis B virus coreantigen protein and inserted therein or attached thereto an amino acidsequence comprising an epitope, wherein the epitope is derived from anextracellular portion of a tumor-associated antigen associated with thesurface of a tumor cell. Preferably, the tumor-associated antigen isexpressed in a limited number of specific tissues and/or organs undernormal conditions and is aberrantly expressed in tumor tissues. In aparticularly preferred embodiment, the protein is capable of eliciting ahumoral immune response directed against the tumor-associated antigen inassociation with the surface of a cell when administered in the form ofa virus-like particle without adjuvant to a subject, wherein thetumor-associated antigen is a self-protein in said subject. Preferably,the humoral immune response comprises the generation of antibodies whichexhibit one or more immune effector functions against cells carrying thetumor-associated antigen in its native conformation, wherein preferablythe one or more immune effector functions are selected from the groupconsisting of complement dependent cytotoxicity (CDC),antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependentcell mediated phagocytosis (ADCP), induction of apoptosis, inhibition ofCD40L-mediated signal transduction, and inhibition of proliferation. Ina preferred embodiment, the immune effector function is activation ofeffector cells, such as ADCC. In a preferred embodiment, thetumor-associated antigen is a protein of the claudin family or PLAC1,wherein preferably the protein of the claudin family is selected fromthe group consisting of CLDN18.2 and CLDN6.

In further aspects, the present invention provides a nucleic acidencoding the protein of the present invention and a vector comprisingthe nucleic acid of the present invention.

In another aspect, the present invention relates to a host cellcomprising the nucleic acid or the vector of the present invention.

In a further aspect, the present invention provides a virus-likeparticle comprising multiple copies of the protein of the presentinvention. It is particularly preferred that the virus-like particle iscapable of eliciting a humoral immune response directed against thetumor-associated antigen in association with the surface of a cell whenadministered without adjuvant to a subject, wherein the tumor-associatedantigen is a self-protein in said subject.

The present invention further provides an immunogenic compositioncomprising the protein, the nucleic acid, the vector, the host cell, orthe virus-like particle of the present invention and a pharmaceuticallyacceptable diluent, carrier, and/or excipient. Preferably, theimmunogenic composition of the present invention is for eliciting ahumoral immune response against the tumor-associated antigen inassociation with the surface of a cell in a subject, wherein thetumor-associated antigen is a self-protein in said subject. It isparticularly preferred that the immunogenic composition is free ofadjuvants.

In a further aspect, the present invention provides the protein, thenucleic acid, the vector, the host cell, the virus-like particle, or theimmunogenic composition of the present invention for prophylactic and/ortherapeutic treatment of tumors.

In a further aspect, the present invention provides a method foreliciting a humoral immune response against a tumor-associated antigenin a subject, wherein the tumor-associated antigen is a self-protein insaid subject, said method comprising administering to said subject theprotein, the nucleic acid, the vector, the host cell, the virus-likeparticle, or the immunogenic composition of the present invention,wherein said subject is afflicted with a tumor or is at risk ofdeveloping a tumor, said tumor being characterized by association of thetumor-associated antigen with the surface of a tumor cell.

In another aspect, the present invention provides a method for breakingself-tolerance towards a tumor-associated antigen in a subject, saidmethod comprising administering to said subject the protein, the nucleicacid, the vector, the host cell, the virus-like particle, or theimmunogenic composition of the present invention, preferably withoutadjuvants.

In another aspect, the present invention provides a method for treatingand/or preventing a tumor in a subject, said method comprisingadministering to said subject the protein, the nucleic acid, the vector,the host cell, the virus-like particle, or the immunogenic compositionof the present invention, preferably without adjuvants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of the HBcAg expression cassettes(HBcAg backbones).

The fusion proteins consist of an amino-terminally andcarboxy-terminally localized region of the HBcAg protein, wherein in allof the HBcAg backbones parts of the major immunodominant region (MIR) ofHBcAg may be replaced by specific antigen epitopes (epitope). Forincreasing the flexibility during assembly into VLPs and an increasedvariance of epitope conformations the epitope inserted into the MIR maybe flanked by glycine linkers (G₄SG₄; SEQ ID NO: 24). All constructswith the exception of HBcAg Del 79-80 linker and HBcAg Del 79-80 carryrestriction sites SalI and SpeI for additional insertion of epitopes atthe amino-terminus of HBcAg. At the carboxy-terminus of the constructs aHis-tag (black box) consisting of six histidines has been incorporatedfor purification under denaturing conditions. The His-tag is separatedfrom the HBcAg carboxy-terminus by a short linker (amino acid sequenceGGS). The available restriction sites for cloning and modificationpurposes are indicated.

FIG. 2: Validation of the assembly competence for selected HBcAg fusionproteins.

In vitro assembled and purified HBcAg VLPs have been analyzed usingnative agarose gel electrophoresis or negative contrast transmissionelectron microscopy (TEM). The analysis is exemplarily shown for thechimeric HBcAg VLPs HBcAg Del 79-80 linker CLDN18.2-EC1 short(CLDN18.2-linker), HBcAg Del 79-80 CLDN18.2-EC1 short (CLDN18.2), andthe truncated variant of HBcAg wild-type (HBcAgΔ; SEQ ID NO: 79) ascontrol. The indicated black bar within the TEM images corresponds to alength of 200 nm.

FIG. 3A-3C: Indirect immunofluorescence analysis for determination ofthe immunoreactivity of the antisera after immunization of Balb/c miceor NZW rabbits.

FIG. 3A) Chinese hamster ovary cells (CHO cells, ATCC No. CCL-61) havebeen co-transfected with eGFP-N3 (GFPmut1 variant) in combination withrabbit or mouse CLDN18.2 and CLDN18.1, respectively, incubated for 24hours, subsequently fixed with 4% paraformaldehyde (PFA) and afterwardspermeabilized with 0.2% saponin. Incubation with diluted (1:100)polyclonal rabbit serum 5 (Immunogen used for the generation of rabbitserum #5: HBcAg Del 79-80 linker CLDN18.2-EC1 short VLPs+Freund'sadjuvants) was carried out for one hour. A CY3-conjugatedgoat-anti-rabbit IgG(H+L) monoclonal antibody has been used as secondaryantibody at a dilution of 1:200 and has been added for 30 minutes. DAPIat a dilution of 1:10000 has been used for staining the cell nuclei.FIG. 3B) The IF analysis has been performed as described under FIG. 3A).For the detection of CLDN18.2 the diluted (1:100) polyclonal mouseantiserum 1/4 (Immunogen used for the generation of antiserum 1/4: HBcAgDel 79-80 linker CLDN18.2-EC1 short VLPs without adjuvants) has beenused. A CY3-conjugated goat-anti-mouse IgG(H+L) monoclonal antibody hasbeen used as secondary antibody at a dilution of 1:200. FIG. 3C) CHOcells have been co-transfected with eGFP-TES85 (localized within thecell nucleus) and human PLAC1 and have been fixed with 4% PFA after 24hours of cultivation. The diluted (1:500) rabbit antiserum PLAC1 #9 hasbeen used as the polyclonal antiserum for detection of PLAC1 (Immunogenused for the generation of antiserum PLAC1 #9: HBcAg Del 79-80 linkerPLAC1 3^(rd) Loop A VLPs+Freund's adjuvants) and PLAC1 #10 (Immunogenused for the generation or antiserum PLAC1 #10: HBcAg Del 79-80 linkerPLAC1 3^(rd) Loop A VLPs without adjuvants), respectively. ACY3-conjugated goat-anti-rabbit IgG(H+L) monoclonal antibody has beenused as a secondary antibody at a dilution of 1:200.

FIG. 4: FACS analysis of the immunoreactivity of rabbit antisera afterimmunization with human CLDN18.2 epitope carrying chimeric HBcAg VLPs.

1×10⁵ NUG-C4 cells endogenously expressing human CLDN18.2 (hsCLDN18.2)have been used for FACS analysis. The cells have been incubated for onehour with diluted (1:50) rabbit serum. After a washing step, incubationwith a diluted (1:100) Alexa647-labeled goat-anti-rabbit IgG(H+L)monoclonal secondary antibody for half an hour has been carried out. Thehistogram overlays of the fluorescent signals for rabbitpre-immunization serum (white) and final sera (gray and black,respectively) are shown. All of the rabbits have been immunized withHBcAg Del 79-80 linker CLDN18.2-EC1 short VLPs. Rabbit 3 has beenimmunized without addition of adjuvants, whereas rabbits 4 and 5 havebeen administered Freund's adjuvant and rabbit 6 has been administeredthe adjuvant Montanide ISA 720 which has been approved for clinicalapplications.

FIG. 5A-5B: Analysis of the cytotoxic effector functions of the CLDN18.2directed polyclonal antisera after active immunization.

FIG. 5A) Luciferase based CDC assay. CHO cells stably expressing humanCLDN18.2 (hsCLDN18.2) or the isoform claudin 18.1 (hsCLDN18.1) have beentransfected with in vitro transcribed (IVT) RNA coding for luciferase 24hours before the assay. Subsequently, the cells have been incubated for30 minutes with the indicated polyclonal antisera before active orheat-inactivated human serum has been added for 30 minutes. Afteraddition of a luciferase containing buffer, the percentage of killedcells has been calculated (after subtraction of background luminescenceand in comparison to cells which have been incubated with active serumbut without addition of antiserum). FIG. 5B) Luciferase based ADCCassay. One day before the assay, NUG-C4 cells endogenously expressinghsCLDN18.2 have been transfected with luciferase IVT-RNA. Subsequently,the cells have been incubated with the indicated polyclonal antisera for30 minutes before isolated human Peripheral Blood Mononuclear Cells(PBMC) as effector cells have been added. After 5 hour incubation, aluciferin-containing buffer has been added and the percentage of killedcells has been calculated (after subtraction of the backgroundluminescence and in comparison to cells which have been incubated withPBMCs but without addition of antiserum). The rabbits 3 to 6 as well asthe mice 1/4, 6/3, and 6/4 have been immunized with HBcAg Del 79-80linker CLDN18.2-EC1 short VLPs (CLDN18.2-Linker). Rabbit 3 and mouse 1/4received the immunogen without addition of adjuvants, whereas rabbits 4and 5 received Freund's adjuvant, rabbit 6 Montanide ISA 720, and mice6/3 and 6/4 Abisco100. The antisera of rabbit 2 which has been immunizedwith C-terminally truncated HBcAg wild-type VLPs (HBcAgΔ), and of mouse11/2 which has been administered a KLH-conjugated peptide of CLDN18.2which sequence was identical to the epitope inserted into HBcAg, havebeen used as controls.

FIG. 6A-6B: Analysis of the anti-proliferative effector functions of thePLAC1 directed polyclonal antisera after active immunization.

FIG. 6A) Inhibition of proliferation of PLAC1 expressing human breastcancer epithelium MCF-7 cells (ATCC No. HTB-22). 5000 cells per wellhave been plated in 10% FCS-containing medium and incubated for 72 hourswith diluted (1:100 or 1:1000) polyclonal PLAC1-directed antisera.Subsequently, a BrdU-based proliferation assay using the Delfia cellproliferation kit has been carried out. The proliferation rate (in %)has been calculated with respect to a medium control (=100%). Apolyclonal anti-CLDN18.2 antiserum which has been generated by activeimmunization (anti-CLDN18.2) as well as a monoclonal anti-myc antibodyhave been used as control sera. FIG. 6B) No inhibition of proliferationof PLAC1-negative MelHO cells. The procedure has been carried out asdescribed under A). HBcAg Del 79-80 linker PLAC1 3^(rd) Loop A (serum #9with adjuvants, serum #10 without adjuvants) as well as HBcAg Del 79-80PLAC1 3^(rd) Loop B (serum #11 with adjuvants) have been used asimmunogens for active anti-PLAC1 immunization.

FIG. 7A-7B: Sequences of the HBcAg backbones.

The nucleic acid and amino acid sequences of the various HBcAg backbonesare shown in one-letter-code. The numbering of the sequence is indicatedleft and right, respectively, of the sequence.

indicates the amino-terminal and carboxy-terminal HBcAg domains,respectively;

indicates inserted glycine linkers (italics, bold) and

indicates the carboxy-terminally localized His-tag (bold).

FIG. 8A-8D: Sequences of the chimeric HBcAg expression constructs whichhave been used for the generation of epitope carrying VLPs.

Nucleic acid and amino acid sequences of the various chimeric HBcAgexpression constructs are shown in one-letter-code. The numbering of thesequence is indicated left and right, respectively, of the sequence.

indicates the amino-terminal and carboxy-terminal HBcAg domains,respectively;

indicates inserted glycine linkers (italics, bold) and

indicates the carboxy-terminally localized His-tag (bold). The aminoacid sequences of the inserted CLDN18.2 and PLAC1 epitopes,respectively, are depicted in bold and are underlined.

FIG. 9A-9D: Prophylactic vaccination with CLDN18.2 epitope carryingchimeric HBcAg VLPs (HBcAg Del 79-80 linker CLDN18.2-EC1 short-VLPs)confers partial protection in an immunocompetent syngeneic mouse tumormodel

Macroscopic analysis of lungs derived from mice vaccinated with HBcAgDel 79-80 linker CLDN18.2-EC1 short-VLPs (CLDN-Link) revealed a smallernumber of metastatic nodules as compared to HBcAg-VLPs comprising aC-terminally truncated protein (amino acids 1-150) (HBcAgΔ) or PBScontrol groups (FIG. 9A) and significantly lower lung weights close tothose of mice not challenged with tumor cells (FIG. 9B). The percentageof cancerous tissue area per whole lung section as calculated aftervisualizing CT26-CLDN18.2 pulmonary metastases by IHC-staining forCLDN18.2 was significantly (p<0.05) smaller as compared to micevaccinated with HBcAgΔ-VLPs or PBS control groups (FIG. 9C, FIG. 9D).

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise. For example, if in a preferredembodiment the protein of the present invention comprises a linkersequence flanking the epitope sequence and in another preferredembodiment the protein of the present invention comprises a peptide tag,it is a contemplated preferred embodiment of the protein of the presentinvention that the protein comprises a linker sequence flanking theepitope sequence and a peptide tag.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kölbl, Eds.,Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, cellbiology, immunology, and recombinant DNA techniques which are explainedin the literature in the field (cf., e.g., Molecular Cloning: ALaboratory Manual, 2^(nd) Edition, J. Sambrook et al. eds., Cold SpringHarbor Laboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step or group of members, integers orsteps but not the exclusion of any other member, integer or step orgroup of members, integers or steps. The terms “a” and “an” and “the”and similar reference used in the context of describing the invention(especially in the context of the claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. Recitation of ranges of values hereinis merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range. Unlessotherwise indicated herein, each individual value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”),provided herein is intended merely to better illustrate the inventionand does not pose a limitation on the scope of the invention otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element essential to the practice of theinvention.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

DEFINITIONS

In the following, definitions will be provided which apply to allaspects of the present invention.

Hepatitis B virus (HBV) is a member of the Hepadnavirus family. Thevirus particle consists of an outer lipid envelope and an icosahedralnucleocapsid core composed of the hepatitis B virus core antigenprotein. The genome of HBV is made of circular DNA. There are four knowngenes encoded by the genome called C, X, P, and S. The core protein iscoded for by gene C and its start codon is preceded by an upstreamin-frame AUG start codon from which the pre-core protein is produced.The protein encoded by gene C is present in at least four differentfunctionally relevant HBV polypeptides: p25 (preC protein), p22(N-terminally cleaved form of the p25), p21 (HBcAg monomer as such), andp17 (HBeAg, N- and C-terminally cleaved form of p25). Of thesepolypeptides only the HBcAg monomer is able to self-assemble into avirus-like particle. Such virus-like particles may contain 180 or 240copies of the HBcAg protein and may be around 30 nm or 34 nm indiameter. The HBcAg protein comprises an N-terminal region which is ableto self-assemble into virus-like particles. The C-terminal limit forC-terminal truncations which are still able to self-assemble into HBcAgparticles was mapped between amino acid residues 139 and 144. TheC-terminal protamine-like arginine-rich domain corresponding to aminoacids 150 to 183 is dispensable for self-assembly. Its function is thebinding of nucleic acids. The so called major immunodominant region(MIR) is localized within a superficial loop around amino acid residues74 to 89 of the HBcAg protein.

The term “hepatitis B virus core antigen protein” or “HBcAg protein” inthe context of the present invention relates to the polypeptide p21 ofany virus belonging to the Hepadnaviridae family, preferably to thehepatitis B virus polypeptide p21 of any hepatitis B virus serotype,such as the serotypes adr, adw, ayr, and ayw, or genotype, such as thegenotypes A, B, C, D, E, F, G, and H. Preferably the hepatitis B viruspolypeptide p21 is derived from the hepatitis B virus serotype ayw.Preferably, the HBcAg protein comprises, preferably essentially consistsof, preferably consists of the amino acid sequence set forth in SEQ IDNO: 1, a variant or portion thereof. The nucleic acid sequence set forthin SEQ ID NO: 2 codes for the amino acid sequence set forth in SEQ IDNO: 1. In the context of the present invention, the term “hepatitis Bvirus core antigen protein” or “HBcAg protein” includes any variantsand/or portions thereof, wherein preferably said variants and/orportions thereof are able to assemble into virus-like particles,preferably into an icosahedral virus-like particle, preferablyconsisting of 180 or 240 copies of HBcAg subunits. Although the C geneis the most conserved amongst the HBV genes, numerous amino acidsubstitutions have been identified for the HBcAg protein. Thus, the term“hepatitis B virus core antigen protein variant” includes, inparticular, any of the naturally occurring variants of the HBcAgprotein. Said term also includes any synthetically generated variantswhich are not naturally occurring and are able to assemble intovirus-like particles.

The term “virus-like particle” or “VLP” refers to an empty virus capsid,which is formed by self-assembly of envelope and/or capsid proteins frommany viruses, including HIV-1, rubella virus, human papilloma virus,Semliki Forest virus, RNA phages, and Hepadnaviridae such as hepatitis Bvirus. The virus-like particles resemble the virus from which they werederived, but lack any viral nucleic acid and therefore, are notinfectious. The virus-like particles of the present invention comprisechimeric hepatitis B virus core antigen proteins. The virus-likeparticles of the invention are non-infectious because they assemblewithout incorporating genetic material. The term “chimeric hepatitis Bvirus core antigen protein” refers to a protein that comprises ahepatitis B virus core antigen protein or a portion thereof and an aminoacid sequence derived from a protein other than a hepatitis B virus coreantigen protein, such as an amino acid sequence comprising an epitopewhich is derived from a tumor-associated antigen. In a preferredembodiment of the present invention, the virus-like particle comprises aHBcAg protein derived from a hepatitis B virus as a carrier for theintegration of heterologous epitopes. However, HBcAg genes from anyother Orthohepadnavirus or Avihepadnavirus can also be used.

The term “portion” refers to a fraction. With respect to a particularstructure such as an amino acid sequence or protein the term “portion”thereof may designate a continuous or a discontinuous fraction of saidstructure. Preferably, a portion of an amino acid sequence comprises atleast 1%, at least 5%, at least 10%, at least 20%, at least 30%,preferably at least 40%, preferably at least 50%, more preferably atleast 60%, more preferably at least 70%, even more preferably at least80%, and most preferably at least 90% of the amino acids of said aminoacid sequence. Preferably, if the portion is a discontinuous fractionsaid discontinuous fraction is composed of 2, 3, 4, 5, 6, 7, 8, or moreparts of a structure, each part being a continuous element of thestructure. For example, a discontinuous fraction of an amino acidsequence may be composed of 2, 3, 4, 5, 6, 7, 8, or more, preferably notmore than 4 parts of said amino acid sequence, wherein each partpreferably comprises at least 5 continuous amino acids, at least 10continuous amino acids, preferably at least 20 continuous amino acids,preferably at least 30 continuous amino acids of the amino acidsequence. The term “part” refers to a continuous element. For example, apart of a structure such as an amino acid sequence or protein refers toa continuous element of said structure. A portion or a part of astructure preferably comprises one or more functional properties of saidstructure. For example, a portion or a part of an epitope is preferablyimmunologically equivalent to the epitope it is derived from.

The term “portion thereof” in the context of the HBcAg protein refers toa portion of the HBcAg protein which comprises at least 30, preferably50, more preferably 80, more preferably 90, more preferably 100, evenmore preferably 110, even more preferably 120, even more preferably 130or more amino acids of the HBcAg protein. The term “portion” alsoincludes a discontinuous portion of the amino acid sequence of the HBcAgprotein. For example, a HBcAg portion of 138 amino acids may consist ofamino acids 1 to 75 and 82 to 144 of the HBcAg protein as set forth inSEQ ID NO: 1 or amino acids corresponding to said amino acids of SEQ IDNO: 1. It is preferred that the discontinuity lies within the regioncorresponding to the MIR, e.g., the region around amino acids 74 to 89of the HBcAg protein. It is preferred that the portion of HBcAg is ableto assemble into virus-like particles. In the case of a discontinuousportion of the HBcAg protein it is preferred that said discontinuousportion is able to assemble into virus-like particles, if the parts ofthe discontinuous portion are joined together in their natural order.This attachment may be direct or by a linker, for example, by an aminoacid sequence comprising an epitope sequence.

The phrase “the protein is able to assemble into virus-like particles”or similar formulations mean that a plurality of the protein (and notjust a single copy of the protein) is able to assemble into virus-likeparticles. In this context, the assembled virus-like particle does notnecessarily have to assume the native HBcAg virus-like particlestructure, i.e., having 180 or 240 subunits in an icosahedral shape, butany structure that is similar to any virus-like particle structure, forexample, a virus-like particle composed of 30, 50, 70, 90, or 150subunits that may, for example, exhibit an irregular shape, as long asthe virus-like particle is stable to a reasonable extend. For example,the virus-like particle should be stable enough to be formulated into apharmaceutical composition and to be administered to a patient. Theskilled person can readily determine whether a protein is able toassemble into virus-like particles. For example, the protein may beexpressed in a heterologous expression system. The assembly of thevirus-like particles occurs within the cytoplasm of the expression host.The cells are harvested, for example, by tangential flow filtration, andlyzed, e.g., using a microfluidizer. The cell debris is removed and thesoluble lysate is assayed for the presence of virus-like particles. Forexample, the virus-like particles may be concentrated and pre-purifiedby ultrafiltration, hydrophobic interaction, hydroxyapatite or sepharoseblue chromatography. The virus-like particles may further be purified byanion exchange chromatography, size exclusion chromatography and/orultrafiltration. The concentrated and purified virus-like particles maythen be detected by negative staining transmission electron microscopy,native agarose gel electrophoresis, asymmetric flow-field-flowfractionation (AF4) combined with dynamic light scattering (DLS), and/orcapture ELISA using a conformation specific monoclonal antibody.

An amino acid sequence (first amino acid sequence) “inserted into”another amino acid sequence (second amino acid sequence) or an aminoacid sequence “inserted therein” means that the first amino acidsequence is integrated into the second amino acid sequence in betweentwo amino acids of the primary structure of the second amino acidsequence. Preferably, the first and the second amino acid sequences areconnected by peptide bonds. One example of an inserted amino acidsequence in the context of the present invention is the insertion of anepitope sequence, for example, derived from a tumor-associated antigen,between two amino acids within the MIR of the HBcAg protein and/orbetween two amino acids within the N-terminus of the HBcAg protein asshown in FIG. 1, e.g., between the SalI and SpeI restriction sites. Inthe context of the present invention, a first amino acid sequenceinserted into a second amino acid sequence may also mean that the firstamino acid sequence replaces one or more, such as 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more, amino acids of the second amino acid sequence.

An amino acid sequence (first amino acid sequence) is meant to be“attached to” another amino acid sequence (second amino acid sequence)if the first amino acid sequence is attached to any amino acid of thesecond amino acid sequence, for example, by chemical cross-linking or apeptide bond. If an amino acid sequence is connected to theamino-terminal or the carboxy-terminal amino acid of the second aminoacid sequence it is attached to the second amino acid sequence. Avariety of cross linkers are commercially available from major supplierssuch as Pierce, Molecular Probes, and Sigma. Homo-bifunctional reagents,specifically recting with primary amine groups (i.e., ε-amino groups oflysine residues) may be used. They are soluble in aqueous solvents andcan form covalent bond. Furthermore, homo-bifunctional imidoesters withvarying lengths of spacer arms between their reactive end groups, suchas dimethyl adipimidate (DMA), dimethyl suberimidate (DMS), and dimethylpimelimidate (DPM) with spacer arm of, for example, 8.6 Å, 11 Å, or 9.2Å may be used. Furthermore, also reversible homobifunctional crosslinkers may be used such as N-hydroxysuccinimide (NHS) esters, such asdithiobis(succinimidylpropionate) (DSP) ordithiobis(sulfosuccinimidylpropionate) DTSSP. Alternatively, aheterobifunctional cross linker may be used, for example, a cross linkerwith one amine-reactive end and a sulfhydryl-reactive moiety at theother. For example, hetero-bifunctional cross linkers with an NHS esterat one end and an SH-reactive groups, such as meleimides or pyridyldisulfides, can be used. Hetero-bifunctional reagents containing aphotoreactive group, such as Bis[2-(4-azidosalicylamido)ethyl)]disulfide BASED, may also be used.

An amino acid sequence (first amino acid sequence) is meant to “replace”(an) amino acid(s) or another amino acid sequence (second amino acidsequence) if the amino acid(s) or the second amino acid sequence is(are) removed completely and instead the first amino acid sequence isplaced at the position where the amino acid(s) or the second amino acidsequence was (were) located.

Residues in two or more polypeptides are said to “correspond” to eachother if the residues occupy an analogous position in the polypeptidestructures. As is well known in the art, analogous positions in two ormore polypeptides can be determined by aligning the polypeptidesequences based on amino acid sequence or structural similarities. Suchalignment tools are well known to the person skilled in the art and canbe, for example, obtained on the World Wide Web, e.g., ClustalW(www.ebi.ac.uk/clustalw) or Align(http://www.ebi.ac.uk/emboss/align/index.html) using standard settings,preferably for Align EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0,Gap Extend 0.5. Those skilled in the art understand that it may benecessary to introduce gaps in either sequence to produce a satisfactoryalignment. Residues in two or more polypeptide sequences are said to“correspond” if the residues are aligned in the best sequence alignment.The “best sequence alignment” between two polypeptides is defined as thealignment that produces the largest number of aligned identicalresidues. The “region of best sequence alignment” ends and, thus,determines the metes and bounds of the length of the comparison sequencefor the purpose of the determination of the similarity score, if thesequence similarity, preferably identity, between two aligned sequencesdrops to less than 30%, preferably less than 20%, more preferably lessthan 10% over a length of 10, 20, or 30 amino acids.

For the purposes of the present invention, “variants” of a protein orpeptide or of an amino acid sequence comprise amino acid insertionvariants, amino acid addition variants, amino acid deletion variantsand/or amino acid substitution variants. Amino acid deletion variantsthat comprise the deletion at the N-terminal and/or C-terminal end ofthe protein are also called N-terminal and/or C-terminal truncationvariants.

Amino acid insertion variants comprise insertions of single or two ormore amino acids in a particular amino acid sequence. In the case ofamino acid sequence variants having an insertion, one or more amino acidresidues are inserted into a particular site in an amino acid sequence,although random insertion with appropriate screening of the resultingproduct is also possible.

Amino acid addition variants comprise amino- and/or carboxy-terminalfusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50,or more amino acids. For example, in the context of the presentinvention, a HBcAg protein comprising a carboxy-terminally fused peptidetag such as a His-tag is considered a HBcAg addition variant.

Amino acid deletion variants are characterized by the removal of one ormore amino acids from the sequence, such as by removal of 1, 2, 3, 5,10, 20, 30, 50, or more amino acids. The deletions may be in anyposition of the protein. In the context of the HBcAg protein used in thepresent invention, a preferred amino acid deletion variant of HBcAg is adeletion within the MIR region, e.g., within the amino acids aroundpositions 74 to 89 of SEQ ID NO: 1 or corresponding amino acids. It isalso preferred in the context of the present invention that the HBcAgprotein is C-terminally truncated, i.e., has a C-terminaldeletion/truncation. Preferably, said C-terminal truncation extends fromthe C-terminus to and including any amino acid down to the amino acid atposition 140 in the amino acid sequence as set forth in SEQ ID NO: 1 ora corresponding amino acid sequence. A C-terminal truncation of aprotein at amino acid position 140 means that the amino acids atposition 140 to the C-terminus of the full-length protein are missing inthe C-terminally truncated protein, i.e., that the C-terminallytruncated protein ends with amino acid 139 (including amino acid 139).

Amino acid substitution variants are characterized by at least oneresidue in the sequence being removed and another residue being insertedin its place. Preference is given to the modifications being inpositions in the amino acid sequence which are not conserved betweenhomologous proteins or peptides and/or to replacing amino acids withother ones having similar properties. Preferably, amino acid changes inprotein variants are conservative amino acid changes, i.e.,substitutions of similarly charged or uncharged amino acids. Aconservative amino acid change involves substitution of one of a familyof amino acids which are related in their side chains. Naturallyoccurring amino acids are generally divided into four families: acidic(aspartate, glutamate), basic (lysine, arginine, histidine), non-polar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), and uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine) amino acids.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids.

Preferably the degree of similarity, preferably identity between a givenamino acid sequence and an amino acid sequence which is a variant ofsaid given amino acid sequence, e.g., between the preferred HBcAgsequence set forth in SEQ ID NO: 1 and the HBcAg variant or between thepreferred tumor-associated antigen sequences, for example, set forth inSEQ ID NOs: and the tumor-associated antigen variants, will be at least60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. The degree ofsimilarity or identity is given preferably for a region of at leastabout 20, at least about 40, at least about 60, at least about 80, atleast about 100, at least about 120, at least about 140 or 160 aminoacids. In preferred embodiments, the degree of similarity or identity isgiven for the entire length of the reference amino acid sequence. Thealignment for determining sequence similarity, preferably sequenceidentity can be done with art known tools, preferably using the bestsequence alignment, for example, using Align, using standard settings,preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend0.5. It is to be understood that in preferred embodiments the HBcAgvariant is a deletion variant and/or a truncation variant, for example,carrying a deletion within the MIR or a truncation at thecarboxy-terminus. Furthermore, in particularly preferred embodiments thevariants are naturally occurring variants. Preferred examples of theHBcAg protein variants, if SEQ ID NO: 1 is used as reference sequence,comprise mutations at one or more of positions.

The proteins and nucleic acid sequences of the present invention alsocomprise variants of the proteins of the present invention and of thenucleic acid sequences of the present invention. The above definitionfor protein variants also applies correspondingly to nucleic acidsequence variants.

The protein and nucleic acid sequence variants described herein mayreadily be prepared by the skilled person, for example, by recombinantDNA manipulation. The manipulation of DNA sequences for preparingproteins and peptides having substitutions, insertions or deletions, isdescribed in detail in Sambrook et al. (1989), for example. Furthermore,the peptides and amino acid variants described herein may be readilyprepared with the aid of known peptide synthesis techniques such as, forexample, by solid phase synthesis and similar methods.

The proteins and peptides described herein may be derivatives ofproteins and peptides. According to the invention, “derivatives” ofproteins and peptides are modified forms of proteins and peptides. Suchmodifications include any chemical modification and comprise single ormultiple substitutions, deletions and/or additions of any moleculesassociated with the protein or peptide, such as carbohydrates, lipidsand/or proteins or peptides. The term “derivative” also extends to allfunctional chemical equivalents of said proteins and peptides.Preferably, a modified peptide, i.e., a derivative peptide, exhibitsincreased stability and/or increased immunogenicity.

According to the invention, a variant, derivative, portion, part, orfragment of a peptide or protein or of a nucleic acid or amino acidsequence preferably has a functional property of the peptide or proteinor the nucleic acid or amino acid sequence, respectively, from which ithas been derived. Such functional properties comprise immunologicalproperties such as the interaction with antibodies, the interaction withother peptides or proteins, and the assembly into virus-like particles.

The term “immunologically equivalent” means that the immunologicallyequivalent amino acid sequence exhibits the same or essentially the sameimmunological properties and/or exerts the same or essentially the sameimmunological effects, e.g., with respect to the type of theimmunological effect such as induction of a humoral and/or cellularimmune response, the strength and/or duration of the induced immunereaction, or the specificity of the induced immune reaction. In thecontext of the chimeric HBcAg protein of the invention, the term“immunologically equivalent” is preferably used with respect to theimmunological effects or properties of the epitope derived from atumor-associated antigen which is comprised by the chimeric HBcAgprotein. A particular immunological property is the ability to bind toantibodies and, where appropriate, generate an immune response,preferably by stimulating the generation of antibodies. For example, anamino acid sequence is immunologically equivalent to a reference aminoacid sequence if said amino acid sequence when exposed to the immunesystem of a subject induces an immune reaction, preferably antibodies,having a specificity of reacting with the reference amino acid sequence,such as the reference amino acid sequence forming part of atumor-associated antigen.

In the context of the present invention, the terms “tumor-associatedantigen” or “tumor antigen” relate to proteins that are under normalconditions specifically expressed in a limited number of tissues and/ororgans or in specific developmental stages, for example, thetumor-associated antigen may be under normal conditions specificallyexpressed in stomach tissue, preferably in the gastric mucosa, inreproductive organs, e.g., in testis, in trophoblastic tissue, e.g., inplacenta, or in germ line cells, and are expressed or aberrantlyexpressed in one or more tumor tissues. In this context, “a limitednumber” preferably means not more than 3, more preferably not more than2. The expression of tumor-associated antigens is reactivated in tumortissues irrespective of the origin of the tumor, i.e., the tissue ororgan the tumor is originated/derived from. The tumor-associatedantigens in the context of the present invention include, for example,differentiation antigens, preferably cell type specific differentiationantigens, i.e., proteins that are under normal conditions specificallyexpressed in a certain cell type at a certain differentiation stage,cancer/testis antigens, i.e., proteins that are under normal conditionsspecifically expressed in testis and sometimes in placenta, and germline specific antigens. In the context of the present invention, thetumor-associated antigen is preferably associated with the cell surfaceof a tumor cell and is preferably not or only rarely expressed in normaltissues. Preferably, the tumor-associated antigen or the aberrantexpression of the tumor-associated antigen identifies tumor cells,preferably cancerous cells. In the context of the present invention, thetumor-associated antigen that is expressed by a tumor cell in a subject,e.g., a patient suffering from a tumorigenic disease, is preferably aself-protein in said subject. In preferred embodiments, noautoantibodies directed against the tumor-associated antigen can befound in a detectable level under normal conditions in a subjectcarrying said tumor-associated antigen or such autoantibodies can onlybe found in an amount below a threshold concentration that would benecessary to cause damage to the tissue or cells carrying saidtumor-associated antigen. In preferred embodiments, the tumor-associatedantigen in the context of the present invention is expressed undernormal conditions specifically in a tissue or organ that isnon-essential, i.e., tissues or organs which when damaged by the immunesystem do not lead to death of the subject, or in organs or structuresof the body which are not or only hardly accessible by the immunesystem. Preferably, the amino acid sequence of the tumor-associatedantigen is identical between the tumor-associated antigen which isexpressed in normal tissues and the tumor-associated antigen which isexpressed in tumorigenic tissues. In the context of the presentinvention, the tumor-associated antigen is preferably not a product of amutated tumor suppressor gene or a mutated oncogene or of any othermutated gene, unless this mutation is present in the germ line of thesubject expressing said tumor-associated antigen. In the context of thepresent invention, the tumor-associated antigen is preferably not atumor antigen produced by an oncogenic virus.

Examples for differentiation antigens which ideally fulfill the criteriafor tumor-associated antigens as contemplated by the present inventionas target structures in tumor immunotherapy, in particular, in tumorvaccination are the cell surface proteins of the claudin family, such asCLDN6 and CLDN18.2, and PLAC1. CLDN18.2 is selectively expressed innormal tissues in differentiated epithelial cells of the gastric mucosa,whereas CLDN6 and PLAC1 have been described as placenta-specificexpression product. These differentiation antigens are expressed intumors of various origins as described herein below, and areparticularly suited as target structures for the development of activevaccination strategies in connection with antibody-mediated cancerimmunotherapy due to their selective expression (no expression in atoxicity relevant normal tissue) and localization to the plasmamembrane.

The terms “normal tissue” or “normal conditions” refer to healthy tissueor the conditions in a healthy subject, i.e., non-pathologicalconditions, wherein “healthy” preferably means non-tumorigenic ornon-cancerous.

The term “specifically expressed” means that a protein is essentiallyonly expressed in a specific tissue or organ. For example, atumor-associated antigen specifically expressed in gastric mucosa meansthat said protein is primarily expressed in gastric mucosa and is notexpressed in other tissues or is not expressed to a significant extentin other tissue or organ types. Thus, a protein that is exclusivelyexpressed in cells of the gastric mucosa and to a significantly lesserextent in any other tissue, such as testis, is specifically expressed incells of the gastric mucosa. In some embodiments, the tumor-associatedantigen may also be specifically expressed under normal conditions inmore than one tissue type or organ, such as in 2 or 3 tissue types ororgans, but preferably in not more than 3 different tissue or organtypes. In this case, the tumor-associated antigen is then specificallyexpressed in these organs. For example, if a tumor-associated antigen isexpressed under normal conditions preferably to an approximately equalextent in lung and stomach, said tumor-associated antigen isspecifically expressed in lung and stomach.

The term “self-protein” in relation to a particular subject in thecontext of the present invention means a protein that is encoded by thegenome of said subject and that is under normal conditions, i.e.,non-pathological conditions, optionally expressed in certain normaltissue types or at certain developmental stages of said subject.Preferably, it does not include proteins with acquired mutations. Atumor-associated antigen that is a self-protein in a subject includes atumor-associated antigen that is or was expressed in said subject undernormal conditions in certain tissues or at a certain developmental stageand is abnormally or aberrantly expressed in tumorigenic tissue of saidsubject, preferably in the same form and/or with the same structure. An“autoantibody” is an antibody that reacts with the cells, tissues, ornative proteins of the individual in which it is produced, i.e., whichreacts with self-proteins of said individual.

The term “self tolerance” designates a mechanism, where the body doesnot mount an immune response to self proteins. Normally, self-toleranceis developed early by developmental events within the immune system thatprevent, in particular, the organism's own T cells and B cells fromreacting with the organism's own tissues.

The term “an extracellular portion of a tumor-associated antigen” in thecontext of the present invention refers to a part of a tumor-associatedantigen facing the extracellular space of a cell preferably beingaccessible from the outside of said cell, e.g., by antibodies locatedoutside the cell. Preferably, the term refers to an extracellular loopor a part thereof or any other extracellular part of a tumor-associatedantigen which is preferably specific for said tumor-associated antigen.Preferably, said part comprises at least 5, at least 8, at least 10, atleast 15, at least 20, at least 30, or at least 50 amino acids or more.

The term “tumor-associated antigen associated with the surface of acell” means that the tumor-associated antigen is associated with andlocated at the plasma membrane of said cell, wherein at least a part ofthe tumor-associated antigen faces the extracellular space of said celland is accessible from the outside of said cell, e.g., by antibodieslocated outside the cell. In this context, a part is preferably at least4, preferably at least 8, preferably at least 12, more preferably atleast 20 amino acids. The association may be direct or indirect. Forexample, the association may be by one or more transmembrane domains,one or more lipid anchors, or by the interaction with any other protein,lipid, saccharide, or other structure that can be found on the outerleaflet of the plasma membrane of a cell. For example, atumor-associated antigen associated with the surface of a cell may be atransmembrane protein having an extracellular portion or may be aprotein associated with the surface of a cell by interacting withanother protein that is a transmembrane protein.

The term “epitope derived from a tumor-associated antigen”, means, forexample, an epitope that is a portion or a part of the tumor-associatedantigen, preferably a portion or a part of the tumor-associated antigenwhich is specific for the tumor-associated antigen, or a variant orderivative thereof, preferably a variant or derivative thereof which isimmunologically equivalent. Preferably, it is possible to identify thetumor-associated antigen from which the epitope is derived based on theepitope sequence. In the context of the term “a tumor derived from aspecific tissue” the term “derived from” means “originated from”.

According to the invention, the term “tumor” or “tumor disease” refersto a swelling or lesion formed by an abnormal growth of cells (calledneoplastic cells or tumor cells). By “tumor cell” is meant an abnormalcell that grows by a rapid, uncontrolled cellular proliferation andcontinues to grow after the stimuli that initiated the new growth cease.Tumors show partial or complete lack of structural organization andfunctional coordination with the normal tissue, and usually form adistinct mass of tissue, which may be benign, pre-malignant, ormalignant.

Preferably, a tumor disease according to the invention is a cancerdisease, i.e., a malignant disease, and a tumor cell is a cancer cell.Preferably, a tumor disease is characterized by cells in which anantigen, i.e., a tumor-associated antigen, preferably a tumor-associatedantigen as defined above, is expressed or aberrantly expressed.Preferably, a tumor disease or a tumor cell is characterized by surfaceexpression of a tumor-associated antigen. In a preferred embodiment ofthe present invention, the tumor or cancer cell is identifiable by acell surface associated tumor-associated antigen, such as by CLDN6,CLDN18.2, or PLAC1.

“Aberrant expression” or “abnormal expression” means according to theinvention that expression is altered, preferably increased, compared tothe state in a non-tumorigenic normal cell or a healthy individual,i.e., in an individual not having a disease associated with aberrant orabnormal expression of a certain protein, e.g., a tumor-associatedantigen. An increase in expression refers to an increase by at least10%, in particular at least 20%, at least 50% or at least 100%, or more.In one embodiment, expression is only found in a diseased tissue, whileexpression in a healthy tissue is repressed.

Preferably, a tumor disease according to the invention is cancer,wherein the term “cancer” according to the invention comprisesleukemias, seminomas, melanomas, teratomas, lymphomas, neuroblastomas,gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenalcancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain,cervical cancer, intestinal cancer, liver cancer, colon cancer, stomachcancer, intestine cancer, head and neck cancer, gastrointestinal cancer,lymph node cancer, esophagus cancer, colorectal cancer, pancreas cancer,ear, nose and throat (ENT) cancer, breast cancer, prostate cancer,cancer of the uterus, ovarian cancer, and lung cancer, and themetastases thereof. Examples thereof are lung carcinomas, mammacarcinomas, prostate carcinomas, colon carcinomas, renal cellcarcinomas, cervical carcinomas, or metastases of the cancer types ortumors described above. The term “cancer” according to the inventionalso comprises cancer metastases.

Preferred tumor-associated antigens in the context of the presentinvention are proteins of the claudin family, preferably CLDN6 orCLDN18.2, or PLAC1.

Claudins are a family of proteins that are the most important componentsof tight junctions, where they establish the paracellular barrier thatcontrols the flow of molecules in the intercellular space between cellsof an epithelium. Claudins are transmembrane proteins spanning themembrane 4 times with the N-terminal and the C-terminal end both locatedin the cytoplasm. The first extracellular loop, termed EC1 or ECL1,consists on average of 53 amino acids, and the second extracellularloop, termed EC2 or ECL2, consists of around 24 amino acids. In thecontext of the present invention, the preferred claudins are CLDN6 (SEQID NOs: 3 and 4) and CLDN18.2 (SEQ ID NOs: 5 and 6). CLDN6 and CLDN18.2have been identified as differentially expressed in tumor tissues, withthe only normal tissues expressing CLDN18.2 being stomach and testis andthe only normal tissue expressing CLDN6 being placenta.

For example, CLDN18.2 has been found to be expressed in pancreaticcarcinoma, esophageal carcinoma, gastric carcinoma, bronchial carcinoma,breast carcinoma, and ENT tumors. CLDN18.2 is a valuable target for theprevention and/or treatment of primary tumors, such as gastric cancer,esophageal cancer, pancreatic cancer, lung cancer such as non small celllung cancer (NSCLC), ovarian cancer, colon cancer, hepatic cancer,head-neck cancer, and cancers of the gallbladder, and metastasesthereof, in particular gastric cancer metastasis such as Krukenbergtumors, peritoneal metastasis, and lymph node metastasis. The cellsexpressing CLDN18.2 are preferably cancer cells and are, in particular,selected from the group consisting of tumorigenic gastric, esophageal,pancreatic, lung, ovarian, colon, hepatic, head-neck, and gallbladdercancer cells.

CLDN6 has been found to be expressed, for example, in ovarian cancer,lung cancer, gastric cancer, breast cancer, hepatic cancer, pancreaticcancer, skin cancer, melanomas, head neck cancer, sarcomas, bile ductcancer, renal cell cancer, and urinary bladder cancer. CLDN6 is aparticularly preferred target for the prevention and/or treatment ofovarian cancer, in particular ovarian adenocarcinoma and ovarianteratocarcinoma, lung cancer, including small cell lung cancer (SCLC)and non-small cell lung cancer (NSCLC), in particular squamous cell lungcarcinoma and adenocarcinoma, gastric cancer, breast cancer, hepaticcancer, pancreatic cancer, skin cancer, in particular basal cellcarcinoma and squamous cell carcinoma, malignant melanoma, head and neckcancer, in particular malignant pleomorphic adenoma, sarcoma, inparticular synovial sarcoma and carcinosarcoma, bile duct cancer, cancerof the urinary bladder, in particular transitional cell carcinoma,kidney cancer, in particular renal cell carcinoma including clear cellrenal cell carcinoma and papillary renal cell carcinoma, colon cancer,testicular embryonal carcinoma, and placental choriocarcinoma, and themetastatic forms thereof. In one embodiment, the cancer diseaseassociated with CLDN6 expression is selected from the group consistingof ovarian cancer, lung cancer, metastatic ovarian cancer and metastaticlung cancer. Preferably, the ovarian cancer is a carcinoma or anadenocarcinoma. Preferably, the lung cancer is a carcinoma or anadenocarcinoma, and preferably is bronchiolar cancer such as abronchiolar carcinoma or bronchiolar adenocarcinoma. In one embodiment,the tumor cell associated with CLDN6 expression is a cell of such acancer.

PLAC1 (SEQ ID NOs: 7 and 8) is a placenta-specific gene which isfrequently aberrantly activated and highly expressed in a variety oftumor types, in particular breast cancer. RNAi-mediated silencing ofPLAC1 in MCF-7 and BT-549 breast cancer cells profoundly impairsmotility, migration, and invasion and induces a G1/S cell cycle blockwith nearly complete abrogation of proliferation. Knock down of PLAC1 isassociated with decreased expression of cyclin D1 and reducedphosphorylation of AKT kinase. Moreover, PLAC1 is localized on thesurface of cancer cells and is accessible for antibodies whichantagonize biological functions of this molecule.

PLAC1 has several properties that make it a highly attractive target fortherapeutic antibodies and/or prophylactic and/or therapeuticvaccination. In the case of breast cancer for example, 82% of patientscarry this target. Her2/neu, in contrast, the target of Herceptin, theonly monoclonal antibody (mAb) available for treatment of this cancertype, is overexpressed in only 20-25% of breast cancer patients (Slamon,D. J., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. G., Keith, D.E., Levin, W. J., Stuart, S. G., Udove, J., Ullrich, A. et al. (1989)Science 244, 707-712). For lung cancer and for gastric cancer, in whichPLAC1 is expressed in 42 and 58% of the cases, respectively, there is noapproved mAb treatment so far owing to the lack of appropriate targetsin these cancer types. PLAC1 is involved not only in proliferation butalso cell motility, migration and invasion.

PLAC1 expression has been found in breast cancer, lung cancer, ovariancancer, gastric cancer, prostate cancer, pancreatic cancer, renal cellcancer, hepatic cancer, sarcoma, thyroid cancer, and head and neckcancer. PLAC1 is a particularly valuable target for the preventionand/or treatment of breast cancer, lung cancer, gastric cancer, ovariancancer, hepatocellular cancer, colon cancer, pancreatic cancer,esophageal cancer, head & neck cancer, kidney cancer, in particularrenal cell carcinoma, prostate cancer, liver cancer, melanoma, sarcoma,myeloma, neuroblastoma, placental choriocarcinoma, cervical cancer, andthyroid cancer, and the metastatic forms thereof. In one embodiment, thecancer disease associated with PLAC1 expression is breast cancer or lungcancer, preferably, metastatic cancer in the lung.

The terms “a subject carrying a tumor-associated antigen” or “a subjectexpressing a tumor associated antigen” are used interchangeably and meanthat a tumor-associated antigen is present in a subject, for example, innormal tissues that express the tumor-associated antigen and/or intumorigenic tissues that express or aberrantly express thetumor-associated antigen. The term “a cell carrying a tumor-associatedantigen” preferably means that said cell carries said tumor-associatedantigen on its surface, i.e., that the tumor-associated antigen isassociated with the surface of said cell.

The term “epitope” refers to an antigenic determinant in a molecule,i.e., to the part in a molecule that is recognized by the immune system,for example, that is recognized by an antibody. For example, epitopesare the discrete, three-dimensional sites on an antigen, which arerecognized by the immune system. In the context of the presentinvention, the epitope is preferably derived from a protein, inparticular a self-protein. An epitope of a protein such as atumor-associated antigen preferably comprises a continuous ordiscontinuous portion of said protein and is preferably between 5 and100, preferably between 5 and 50, more preferably between 8 and 30, mostpreferably between 10 and 25 amino acids in length, for example, theepitope may be preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, or 25 amino acids in length. The epitope in the contextof the present invention is derived from a tumor-associated antigen,preferably a tumor-associated antigen which is a self-protein in asubject suffering from a disease associated with expression or aberrantexpression of said tumor-associated antigen, e.g., a tumorigenic diseasesuch as cancer. It is particularly preferred that the epitope in thecontext of the present invention is not a T-cell epitope. Preferably,the epitope in the context of the present invention is a B-cell epitope.The phrase “the epitope comprised by the nucleic acid of the presentinvention or the vector of the present invention” means “the nucleicacid coding for the epitope and being comprised by the nucleic acid ofthe present invention or the vector of the present invention”.

The term “an amino acid sequence comprising an epitope” refers to anamino acid sequence that includes the amino acid sequence(s) of one ormore epitopes and may optionally include other sequences such as linkersequences. If the amino acid sequence comprising an epitope comprisesmore than one epitopes, said epitopes may be identical to or differentfrom each other. Preferably, the epitope is derived from atumor-associated antigen as set forth above. The length of the aminoacid sequence comprising an epitope is preferably such that wheninserted or attached to a HBcAg protein, the chimeric HBcAg protein isstill capable of assembling into virus-like particles. For example, thelength of the amino acid sequence comprising an epitope may be up to 10,up to 20, up to 30, up to 50, up to 100, up to 150, up to 200, up to250, or up to 300 amino acids.

A “linker sequence” is preferably an amino acid sequence connecting twoother amino acid sequences. For example, a part of the HBcAg protein maybe connected with a part of a tumor-associated antigen sequence, e.g.,an epitope sequence, via a linker sequence. A preferred linker sequenceis G₄SG₄ (SEQ ID NO: 24).

The terms “eliciting an immune response” and “inducing an immuneresponse” are used interchangeably in the context of the presentinvention and preferably refer to induction of a humoral immuneresponse. A humoral immune response preferably comprises the generationof antigen-specific antibodies, in particular, of epitope-specificantibodies. In the context of the present invention, the humoral immuneresponse preferably comprises the generation of antibodies directedagainst a tumor-associated antigen, wherein preferably thetumor-associated antigen is a self-protein in the subject in which thehumoral immune response is elicited. Thus, in preferred embodiments, theantibodies generated during the humoral immune response areautoantibodies, preferably directed against the tumor-associated antigenin its native conformation on the surface of a cell, e.g., a tumor cell,preferably a living tumor cell. In the context of the present invention,the antibodies generated during a humoral immune response are preferablycapable of eliciting immune effector functions as described herein.Preferably, said immune effector functions are directed against cellscarrying the tumor-associated antigen from which the epitope is derivedon their surface. For example, the generated antibodies are capable ofmediating ADCC and/or CDC against such cells and/or they directly induceapoptosis in or inhibit proliferation of the cells carrying thetumor-associated antigen on their surface. The terms “eliciting animmune response” and “inducing an immune response” may also refer to theinduction of a cellular immune response and the induction of a cellularas well as a humoral immune response. The immune response, preferablythe humoral immune response, may be protective/preventive/prophylacticand/or therapeutic. “Inducing” may mean that there was no immuneresponse against a particular antigen before induction, but it may alsomean that there was a certain level of immune response against aparticular antigen before induction and after induction said immuneresponse is enhanced. Thus, “inducing the immune response” in thiscontext also includes “enhancing the immune response”. Preferably, afterinducing an immune response in an individual, said individual isprotected from developing a disease such as a cancerous disease or thedisease condition is ameliorated by inducing an immune response. Forexample, an immune response against a tumor-associated antigen may beelicited in a patient having cancer or in a subject being at risk ofdeveloping cancer. Eliciting an immune response in this case may meanthat the disease condition of the patient is ameliorated, that thepatient does not develop metastases, or that the subject being at riskof developing cancer does not develop cancer.

A “cellular immune response” or a “cellular response against an antigen”is meant to include a cellular response directed to cells characterizedby presentation of an antigen with class I or class II MHC. The cellularresponse relates to cells called T-cells or T-lymphocytes which act aseither ‘helpers’ or ‘killers’. The helper T cells (also termed CD4⁺ Tcells) play a central role by regulating the immune response and thekiller cells (also termed cytotoxic T-cells, cytolytic T-cells, CD8⁺T-cells or CTLs) kill diseased cells such as tumor cells, preventing theproduction of more diseased cells.

The term “antibody” refers to a glycoprotein comprising at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds, or an antigen binding portion thereof. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as VH)and a heavy chain constant region. Each light chain is comprised of alight chain variable region (abbreviated herein as VL) and a light chainconstant region. The VH and VL regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each VH and VL is composed of three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies maymediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (Clq) of the classical complement system.

The term “immune effector functions” in the context of the presentinvention includes any functions mediated by components of the immunesystem that result in the inhibition of tumor growth and/or inhibitionof tumor development, including inhibition of tumor dissemination andmetastasis. Preferably, immune effector functions result in killing oftumor cells. Preferably, the immune effector functions in the context ofthe present invention are antibody-mediated effector functions. Suchfunctions comprise complement dependent cytotoxicity (CDC),antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependentcell-mediated phagocytosis (ADCP), induction of apoptosis in the cellscarrying the tumor-associated antigen, for example, by binding of theantibody to a surface antigen, inhibition of CD40L-mediated signaltransduction, for example, by binding of the antibody to the CD40receptor or CD40 ligand (CD40L), and/or inhibition of proliferation ofthe cells carrying the tumor-associated antigen, preferably ADCC and/orCDC. Thus, antibodies that are capable of mediating one or more immuneeffector functions are preferably able to mediate killing of cells byinducing CDC-mediated lysis, ADCC-mediated lysis, apoptosis, homotypicadhesion, and/or phagocytosis, preferably by inducing CDC-mediated lysisand/or ADCC-mediated lysis. Antibodies may also exert an effect simplyby binding to tumor-associated antigens on the surface of a tumor cell.For example, antibodies may block the function of the tumor-associatedantigen or induce apoptosis just by binding to the tumor-associatedantigen on the surface of a tumor cell. For example, antibodies bindingto PLAC1 on the cell surface blocks proliferation of the cells. In apreferred embodiment, the tumor-associated antigen is PLAC1 and theeffector functions exerted by the antibodies induced against PLAC1comprise inhibition of proliferation.

ADCC describes the cell-killing ability of effector cells, in particularlymphocytes, which preferably requires the target cell being marked byan antibody. ADCC preferably occurs when antibodies bind to antigens ontumor cells and the antibody Fc domains engage Fc receptors (FcR) on thesurface of immune effector cells. Several families of Fc receptors havebeen identified, and specific cell populations characteristicallyexpress defined Fc receptors. ADCC can be viewed as a mechanism todirectly induce a variable degree of immediate tumor destruction thatalso leads to antigen presentation and the induction of tumor-directedT-cell responses. Preferably, in vivo induction of ADCC will lead totumor-directed T-cell responses and further host-derived antibodyresponses.

CDC is another cell-killing method that can be directed by antibodies.IgM is the most effective isotype for complement activation. IgG1 andIgG3 are also both very effective at directing CDC via the classicalcomplement-activation pathway. Preferably, in this cascade, theformation of antigen-antibody complexes results in the uncloaking ofmultiple Clq binding sites in close proximity on the C_(H)2 domains ofparticipating antibody molecules such as IgG molecules (C1q is one ofthree subcomponents of complement C1). Preferably these uncloaked C1qbinding sites convert the previously low-affinity C1q-IgG interaction toone of high avidity, which triggers a cascade of events involving aseries of other complement proteins and leads to the proteolytic releaseof the effector-cell chemotactic/activating agents C3a and C5a.Preferably, the complement cascade ends in the formation of a membraneattack complex, which creates pores in the cell membrane that facilitatefree passage of water and solutes into and out of the cell and may leadto apoptosis.

The term “immune effector cells” in the context of the present inventionrelates to cells which exert effector functions during an immunereaction. For example, such cells secrete cytokines and/or chemokines,kill microbes, secrete antibodies, recognize infected or cancerouscells, and optionally eliminate such cells. For example, immune effectorcells comprise T-cells (cytotoxic T-cells, helper T-cells, tumorinfiltrating T-cells), B-cells, natural killer cells, neutrophils,macrophages, and dendritic cells.

The terms “subject” and “individual” are used interchangeably and relateto mammals. For example, mammals in the context of the present inventionare humans, non-human primates, domesticated animals such as dogs, cats,sheep, cattle, goats, pigs, horses etc., laboratory animals such asmice, rats, rabbits, guinea pigs, etc. as well as animals in captivitysuch as animals of zoos. The term “animal” as used herein also includeshumans. The term “subject” may also include a patient, i.e., an animal,preferably a human having a disease, preferably a disease associatedwith expression or aberrant expression of a tumor-associated antigensuch as CLDN18.2, CLDN6, or PLAC1, preferably a tumorigenic disease suchas a cancer. In preferred embodiments, the immune system of the subjectis not compromised or is essentially not compromised. This means thatessential properties of a properly functioning immune system of thesubject are present in the subject. This includes, in particular, thatthe subject is able of producing an immune reaction towards anadministered antigen which is comparable to an immune reaction whichwould be expected from an individual with a normally functioning immunesystem, e.g., with respect to the type of the immune reaction such asinduction of a humoral and/or cellular immune response, the strengthand/or duration of the induced immune reaction, or the specificity ofthe induced immune reaction. Alternatively or additionally, this mayinclude that self-tolerance mechanisms are maintained and present insaid subject. For example, these self-tolerance mechanisms could, e.g.,result in a suppression of an immune reaction against a tumor-associatedantigen which is a self-protein if administered as such.

According to the invention, the term “nucleic acid” comprisesdeoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinationsthereof, and modified forms thereof. The term comprises genomic DNA,cDNA, mRNA, recombinantly produced and chemically synthesized molecules.According to the invention, a nucleic acid may be present as asingle-stranded or double-stranded and linear or covalently circularlyclosed molecule.

In the context of the present invention, the terms “immunogeniccomposition” and “vaccine composition” are used interchangeably andrelate to an antigenic preparation which comprises the protein accordingto the present invention, preferably in the form of a virus-likeparticle. The immunogenic composition may be administered to a subjectin order to stimulate the humoral and/or cellular immune system of saidsubject against one or more antigens, preferably against one or moretumor-associated antigens, which are preferably self-proteins in saidsubject. An immunogenic composition in the context of the presentinvention preferably exerts its immunogenic potential without theaddition of adjuvants and is preferably administered to a subject in anysuitable route in order to elicit a protective and/or therapeutic immunereaction against the antigen, e.g., the tumor-associated antigen fromwhich the epitope is derived which is comprised by the protein of thepresent invention. In a preferred embodiment, the immunogeniccomposition according to the present invention is capable of inducingantibody generation against the epitope derived from a tumor-associatedantigen within the subject administered with said immunogeniccomposition, wherein the tumor-associated antigen is preferably aself-protein within said subject. In other words, in a particularlypreferred embodiment, the immunogenic composition of the presentinvention is capable of eliciting a humoral immune response whichcomprises the generation of autoantibodies against a tumor-associatedantigen which is a self-protein in the subject to which the immunogeniccomposition has been administered.

The term “breaking self-tolerance” refers to any procedure that causesthe immune system of a subject to generate an immune response against aself-protein. Usually, self-proteins are protected from a subject's ownimmune system due to self-tolerance. The immune system recognizesself-proteins as “self” and does not attack such structures. This meansfor tumor-associated antigens, which are often self-proteins, that cellscarrying said tumor-associated antigens are not recognized as foreign ordiseased and thus are not attacked by the immune system due to anexisting self-tolerance towards said antigens. Thus, in the context oftumor therapy, it is desired to break self-tolerance towardstumor-associated antigens.

The term “immunotherapy” relates to a treatment involving activation ofa specific immune reaction. In the context of the present invention,terms such as “protect”, “prevent”, “prophylactic”, “preventive”, or“protective” relate to the prevention or treatment or both of theoccurrence and/or the propagation of a tumor in an individual. The term“immunotherapy” in the context of the present invention preferablyrefers to active tumor immunization or tumor vaccination. A prophylacticadministration of an immunotherapy, for example, a prophylacticadministration of the immunogenic composition of the invention,preferably protects the recipient from the development of tumor growth.A therapeutic administration of an immunotherapy, for example, atherapeutic administration of the immunogenic composition of theinvention, may lead to the inhibition of the progress/growth of thetumor. This comprises the deceleration of the progress/growth of thetumor, in particular a disruption of the progression of the tumor, whichpreferably leads to elimination of the tumor. A therapeuticadministration of an immunotherapy may protect the individual, forexample, from the dissemination or metastasis of existing tumors.

The term “immunization” or “vaccination” describes the process ofadministering antigen to a subject with the purpose of inducing animmune response for therapeutic or prophylactic reasons.

The term “adjuvant” relates to compounds which when administered incombination with an antigen or antigen peptide to an individual prolongsor enhances or accelerates the immune response. The immunogeniccomposition of the present invention preferably exerts its immunogeniceffect without addition of adjuvants. Still, the immunogenic compositionof the present invention may contain any known adjuvant. It is assumedthat adjuvants exert their biological activity by one or moremechanisms, including an increase of the surface of the antigen, aprolongation of the retention of the antigen in the body, a retardationof the antigen release, targeting of the antigen to macrophages,increase of the uptake of the antigen, enhancement of antigenprocessing, stimulation of cytokine release, stimulation and activationof immune cells such as B-cells, macrophages, dendritic cells, T-cellsand unspecific activation of immune cells. Adjuvants comprise aheterogeneous group of compounds such as oil emulsions (e.g., Freund'sadjuvants), mineral compounds (such as alum), bacterial products (suchas Bordetella pertussis toxin), liposomes, and immune-stimulatingcomplexes. Examples for adjuvants are monophosphoryl-lipid-A (MPLSmithKline Beecham). Saponins such as QS21 (SmithKline Beecham), DQS21(SmithKline Beecham; WO 96/33739), QS7, QS17, QS18, and QS-L1 (So etal., 1997, Mol. Cells 7: 178-186), incomplete Freund's adjuvants,complete Freund's adjuvants, vitamin E, montanid, alum, CpGoligonucleotides (Krieg et al., 1995, Nature 374: 546-549), Flt3 ligands(DE 10 2008 061 522), and various water-in-oil emulsions which areprepared from biologically degradable oils such as squalene and/ortocopherol.

Terms such as “increasing” or “enhancing” preferably relate to anincrease or enhancement by about at least 10%, preferably at least 20%,preferably at least 30%, more preferably at least 40%, more preferablyat least 50%, even more preferably at least 80%, and most preferably atleast 100%. These terms may also relate to circumstances, wherein attime zero there is no detectable signal for a certain compound orcondition and at a particular time point later than time zero there is adetectable signal for a certain compound or condition.

The immunogenic composition according to the present invention isgenerally applied in “pharmaceutically acceptable amounts” and in“pharmaceutically acceptable preparations”. Such compositions maycontain salts, buffers, preserving agents, carriers and optionally othertherapeutic agents. “Pharmaceutically acceptable salts” comprise, forexample, acid addition salts which may, for example, be formed by mixinga solution of compounds with a solution of a pharmaceutically acceptableacid such as hydrochloric acid, sulfuric acid, fumaric acid, maleicacid, succinic acid, acetic acid, benzoic acid, citric acid, tartaricacid, carbonic acid, or phosphoric acid. Furthermore, where the compoundcarries an acidic moiety, suitable pharmaceutically acceptable saltsthereof may include alkali metal salts (e.g., sodium or potassiumsalts); alkaline earth metal salts (e.g., calcium or magnesium salts);and salts formed with suitable organic ligands (e.g., ammonium,quaternary ammonium and amine cations formed using counteranions such ashalide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkylsulfonate and aryl sulfonate). Illustrative examples of pharmaceuticallyacceptable salts include, but are not limited to, acetate, adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate,bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate,camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate,clavulanate, cyclopentanepropionate, digluconate, dihydrochloride,dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate,formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate,glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate,hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide,isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate,maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate,mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate,N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate),palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate,phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate,salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate,teoclate, tosylate, triethiodide, undecanoate, valerate, and the like(see, for example, S. M. Berge et al., “Pharmaceutical Salts”, J. Pharm.Sci., 66, pp. 1-19 (1977)).

The term “excipient” when used herein is intended to indicate allsubstances in a pharmaceutical formulation which are not activeingredients such as, e.g., carriers, binders, lubricants, thickeners,surface active agents, preservatives, emulsifiers, buffers, flavoringagents, or colorants.

The immunogenic compositions according to the present invention maycomprise a pharmaceutically acceptable carrier. The term“pharmaceutically acceptable carrier” in the context of the presentinvention relates to one or more compatible solid or liquid fillers ordiluents, which are suitable for an administration to a human. The term“carrier” relates to a natural or synthetic organic or inorganiccomponent which is combined with an active component in order tofacilitate the application of the active component. Preferably, carriercomponents are sterile liquids such as water or oils, including thosewhich are derived from mineral oil, animals, or plants, such as peanutoil, soy bean oil, sesame oil, sunflower oil, etc. Salt solutions andaqueous dextrose and glycerin solutions may also be used as aqueouscarrier compounds.

According to the present invention, the immunogenic composition isadministered in a therapeutically effective amount. A “therapeuticallyeffective amount” relates to an amount which—alone or in combinationwith further dosages—results in a desired reaction or a desired effect.In the case of the therapy of a particular disease or a particularcondition, the desired reaction relates to the inhibition of theprogress of the disease. This comprises the deceleration of the progressof the disease, in particular a disruption of the progression of thedisease. The desired reaction for a therapy of a disease or a conditionmay also be the retardation of the occurrence or the inhibition of theoccurrence of the disease or the condition. An effective amount of thecomposition according to the present invention is dependent on thecondition or disease, the severity of the disease, the individualparameters of the patient, including age, physiological condition,height, and weight, the duration of the treatment, the type of anoptionally accompanying therapy, the specific administration route, andsimilar factors. In case the reaction of a patient is insufficient withan initial dosage, multiple immunizations or higher dosages (or highereffective dosages which may be achieved by a more localizedadministration route) may be applied. In general, for a treatment or foran induction or increase of an immune reaction in a human preferablydosages of the protein of the present invention, preferably of thevirus-like particle of the present invention, in the range of 0.01 to200 μg/kg body weight, and preferably in the range of 0.1 to 100 μg/kgare formulated and administered. In a preferred embodiment, 50 μg to 2mg, preferably 600 μg of the virus-like particle of the invention isadministered to a human patient having a body weight of about 80 kg.Preferably, this amount is administered three times, preferablyaccording to a standard immunization protocol.

In one embodiment, the immunogenic compositions according to the presentinvention are administered no more than five times, no more than fourtimes, no more than three times, or no more than two times over a periodof 40 days, 30 days, 20 days, 15 days, or 10 days starting with thefirst administration of the immunogenic compositions according to thepresent invention. In one embodiment, the immunogenic compositionsaccording to the present invention are administered three times, or twotimes over a period of 30 days, 20 days, 15 days, or 10 days startingwith the first administration of the immunogenic compositions accordingto the present invention. Preferably, this administration of theimmunogenic compositions according to the present invention is followedby one or more, preferably singular booster administrations usingimmunogenic compositions according to the present invention whichpreferably are given not earlier than 15 days, 20 days, 40 days, 50days, or 60 days after the last administration of the immunogeniccompositions according to the present invention or the last boosteradministration.

The term “expression” is used herein in its broadest meaning andcomprises the production of RNA or of RNA and protein. With respect toRNA, the term “expression” or “translation” relates in particular to theproduction of peptides or proteins. Expression may be transient or maybe stable.

According to the present invention, the term “peptide” comprises oligo-and polypeptides and refers to substances comprising two or more,preferably three or more, preferably four or more, preferably six ormore, preferably eight or more, preferably ten or more, preferably 14 ormore, preferably 16 or more, preferably 21 or more and up to preferably8, 10, 20, 30, 40, or 50, in particular 100 amino acids joint covalentlyby peptide bonds. The term “protein” refers to large peptides,preferably to peptides with more than 100 amino acid residues, but ingeneral the terms “peptides” and “proteins” are synonymous and are usedinterchangeably herein.

DETAILED DESCRIPTION

The present inventors have surprisingly found that it is possible toinduce a humoral immune response in a subject, i.e., to induce asubject's immune system to generate antibodies, in particularautoantibodies, against tumor-associated antigens, wherein the generatedantibodies are capable of recognizing the tumor-associated antigen inits native form on the surface of a cell and of exerting immune effectorfunctions against cells carrying said tumor-associated antigen. Inparticular, the present invention makes use of virus-like particlescomposed of a hepatitis B virus core antigen protein as carrier forepitopes derived from tumor-associated antigens.

For all aspects of the present invention relating to induction of immuneresponses and/or prophylactic and/or therapeutic treatments oftumorigenic diseases, it is to be understood that the immune response isinduced against the tumor-associated antigen from which the epitope isderived that is inserted into or attached to specific locations of theHBcAg protein and that the prophylactic and/or therapeutic treatment ofthe tumorigenic disease is with respect to a tumorigenic diseaseassociated with surface expression of the tumor-associated antigen fromwhich the epitope is derived. For example, if the epitope is derivedfrom CLDN6, the induced immune response and the prophylactic and/ortherapeutic treatment is directed against CLDN6 expressing cells,preferably CLDN6 expressing tumor cells, and against tumorigenicdiseases associated with CLDN6 expression. The same applies for CLDN18.2and PLAC1 and any other tumor-associated antigen. The specific preferredtumor types for the specific tumor-associated antigens are given hereinand apply to all aspects of the present invention.

In one aspect, the present invention provides a protein comprising allor a portion of the amino acid sequence of a hepatitis B virus coreantigen protein and inserted therein or attached thereto an amino acidsequence comprising an epitope, wherein the epitope is derived from anextracellular portion of a tumor-associated antigen associated with thesurface of a tumor cell. Preferably, the tumor-associated antigen isexpressed in a limited number of specific tissues and/or organs undernormal conditions, preferably in not more than 3, more preferably in notmore than 2, most preferably in one specific tissue or organ and isexpressed or aberrantly expressed in tumor tissues.

Preferably, the amino acid sequence comprising the epitope is insertedinto or attached to the amino acid sequence of the hepatitis B viruscore antigen protein or the portion thereof such that the epitopeassumes at least partially its native conformation. Native conformation,in this context, means that the epitope exhibits the same structure asit assumes within its natural environment, i.e., within thetumor-associated antigen it is derived from, under native conditions,i.e., under conditions the tumor-associated antigen is usually found in.The term “partially” in this context may mean that the structure of theepitope within the chimeric HBcAg protein is not necessarily 100%identical to the structure of the epitope within the tumor-associatedantigen, but that at least a significant similarity can be identified.Thus, preferably, antibodies generated against the epitope within theprotein of the present invention are able to recognize and to bind tothe tumor-associated antigen in its native conformation preferably onthe surface of a cell, preferably a tumor cell, preferably a livingtumor cell, preferably a living tumor cell in its natural environment.Preferably, the epitope assumes such conformation that an immuneresponse against cells carrying the tumor-associated antigen is elicitedin a subject expressing the tumor-associated antigen when the protein ofthe invention is administered to said subject, preferably in the form ofa virus-like particle. Preferably said immune response is elicited evenwhen the tumor-associated antigen is a self-protein in said subject. Ina preferred embodiment, the protein of the present invention is capableof eliciting an immune response, preferably a humoral immune response,against the tumor-associated antigen the epitope is derived from in asubject against a self-tolerance towards the tumor-associated antigenexisting in said subject. Preferably, the protein of the presentinvention is capable of eliciting said immune response, preferably inthe form of a virus-like particle, even when administered withoutadjuvant.

In a particularly preferred embodiment of the protein of the presentinvention, said protein is capable of eliciting a humoral immuneresponse directed against the tumor-associated antigen in associationwith the surface of a cell when administered in the form of a virus-likeparticle without adjuvant to a subject, wherein the tumor-associatedantigen is a self-protein in said subject. Preferably, the humoralimmune response comprises the generation of antibodies, preferablyautoantibodies, which exhibit one or more immune effector functions,preferably against cells carrying the tumor-associated antigen in itsnative conformation. Preferably, the one or more immune effectorfunctions are selected from the group consisting of complement dependentcytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity(ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), inductionof apoptosis, inhibition of proliferation, and inhibition ofCD40L-mediated signal transduction, preferably the effector functionsare ADCC and/or CDC.

The skilled person can readily determine whether a protein fulfills theabove criteria. For example, the skilled person may generate a proteinas described above comprising an epitope derived from the extracellularportion of a tumor-associated antigen, such as CLDN6, CLDN18.2, orPLAC1, using, for example, a mouse- or rabbit-specific amino acidsequence. The skilled person may then immunize rabbits with a proteincomprising the rabbit-specific epitope or mice with the proteincomprising the mouse-specific epitope, wherein the protein is preferablyin the form of a virus-like particle and is preferably administeredwithout adjuvants. The skilled person is well aware of immunizationschemes and any of these schemes may be employed. After immunization iscompleted, serum may be harvested from the animals and the serum may betested for antibody-mediated effector functions on cells, preferablytumor cells, endogenously or exogenously expressing the tumor-associatedantigen having the amino acid sequence of the respective species theepitope was derived from and the immunization has been performed in. Forexample, killing of cells carrying the respective tumor-associatedantigen on their surface or inhibition of proliferation of such cellscan be determined. Such methods are exemplarily described in theExamples section of the present invention.

In a particular preferred embodiment, the tumor-associated antigen is aprotein of the claudin family or PLAC1. Preferably, the protein of theclaudin family is selected from the group consisting of CLDN18.2 andCLDN6.

The epitope is preferably between 5 amino acids and the entire length ofa continuous part of the extracellular portion of the tumor-associatedantigen and is preferably specific for said tumor-associated antigen.For example, the epitope may be between 5 and 100, preferably between 5and 50, more preferably between 8 and 30, most preferably between 10 and25 amino acids in length, for example, the epitope may be preferably 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aminoacids in length.

Particularly preferred epitope sequences in the context of the presentinvention are for CLDN6 PMWKVTAFIGNSI (SEQ ID NO: 9), MWKVTAFIGNSIVVA(SEQ ID NO: 10), FIGNSIVVAQVVWE (SEQ ID NO: 11), VVAQVVWEGLWMS (SEQ IDNO: 12), VAQVVWEGLWMSCVVQSTGQMQC (SEQ ID NO: 13), KVTAFIGNSIVVAQVV (SEQID NO: 14), KVTAFIGNSIVVAQ (SEQ ID NO: 15), RDFYNPLVAEAQK (SEQ ID NO:16), DFYNPLVAEAQ (SEQ ID NO: 17), TAHAIIRDFYNPL (SEQ ID NO: 18),DFYNPLVAEAQK (SEQ ID NO: 19), and IRDFYNPLVAEAQKRE (SEQ ID NO: 20), forCLDN18.2 TQDLYNNPVT (SEQ ID NO: 21), DLYNNPVTAVFNYQGL (SEQ ID NO: 45),NNPVTAVFNYQ (SEQ ID NO: 46), VTAVFNYQGL (SEQ ID NO: 47), SCVRESSGF (SEQID NO: 48), VRESSGFT (SEQ ID NO: 49), VRESSGFTE (SEQ ID NO: 50),RGYFTLLGL (SEQ ID NO: 51), ECRGYFTLLGL (SEQ ID NO: 52), AVFNYQGLWRSCVRES (SEQ ID NO: 53), DQWSTQDLYNNPVTAVFNYQ (SEQ ID NO: 54),MDQWSTQDLYNNPVTAVFNYQGL (SEQ ID NO: 55), WRSCVRESSGFTECRGYFTLLGLPAMLQAVR (SEQ ID NO: 56), RIGSMEDSAKANMTLTS (SEQ ID NO: 57),TNFWMSTANMYTGMGGMVQTVQTRYTF (SEQ ID NO: 58), and for PLAC1 VFSEEEHTQVP(SEQ ID NO: 22), VFSEEEHTQV (SEQ ID NO: 23), APQKSPWLTKP (SEQ ID NO:59), QKSPWLTKP (SEQ ID NO: 60), APQKSPWLT (SEQ ID NO: 61), MRVASKSR (SEQID NO: 62), APQKSP (SEQ ID NO: 63), TAQKDEK (SEQ ID NO: 64), SKGTPSK(SEQ ID NO: 65), APQKSPWLTK (SEQ ID NO: 66), QKSPWLTK (SEQ ID NO: 67),SMRVASKSRATAQKDEK (SEQ ID NO: 68), PPNHVQPHAYQFTYRVTE (SEQ ID NO: 69),SMRVASKSKRATAQKDE (SEQ ID NO: 70), SMRVASKSKRATA QKD (SEQ ID NO: 71),SMRVASKSKRATAQK (SEQ ID NO: 72), RVASKSKRATA (SEQ ID NO: 73),YEVFSLSQSSQRPN (SEQ ID NO: 74), EVFSLSQSSQR (SEQ ID NO: 75),IDWFMVTVHPFMLNNDV (SEQ ID NO: 76), IDWFMVTVHPFMLNND (SEQ ID NO: 77),IDWFMVTVHPFMLNN (SEQ ID NO: 78), and variants thereof.

In a preferred embodiment of the protein of the present invention, theepitope comprises, preferably essentially consists of, preferablyconsists of

-   -   (i) an amino acid sequence which is selected from the group        consisting of the amino acid sequences set forth in SEQ ID NOs:        9 to 23 and 45 to 78 of the sequence listing,    -   (ii) an amino acid sequence that is at least at least 60%, 65%,        70%, 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, preferably at least        80%, identical to the amino acid sequence under (i) preferably        over the entire length of the epitope sequence, and is        immunologically equivalent to the amino acid sequence under (i),        or    -   (iii) an amino acid sequence under (i) or (ii) which is        truncated and is immunologically equivalent to the amino acid        sequence under (i). Said truncation may be at the amino-terminus        or at the carboxy-terminus and is preferably not more than 40%,        preferably not more than 30%, preferably not more than 20%, more        preferably not more than 10% of the entire length of the epitope        sequence.

Variants and/or derivatives of these epitopes are also contemplated inthe present invention as long as said variants and/or derivatives areimmunologically equivalent to the specifically disclosed epitopes. Theskilled person will understand that single amino acid substitutions,additions, insertions, and/or deletions within the epitope may not alterthe immunological properties of said epitopes significantly.

In a preferred embodiment of the protein of the present invention, thehepatitis B virus core antigen protein comprises, preferably essentiallyconsists of, preferably consists of an amino acid sequence selected fromthe group consisting of

-   -   (i) the amino acid sequence set forth in SEQ ID NO: 1 or a        portion thereof of at least 50 amino acids, preferably of at        least 60 amino acids, preferably of at least 70 amino acids,        preferably of at least 80 amino acids, preferably of at least 90        amino acids, preferably of at least 100 amino acids, preferably        of at least 110 amino acids, preferably of at least 120 amino        acids, preferably of at least 130 amino acids, preferably of at        least 140 amino acids, or    -   (ii) an amino acid sequence that is at least 60%, 65%, 70%, 80%,        81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, preferably at        least 80% identical, to the amino acid sequence or the portion        thereof under (i) preferably over the entire length of the amino        acid sequence or the portion thereof under (i). Preferably, the        hepatitis B virus core antigen protein used in the present        invention is functionally equivalent to the naturally occurring        hepatitis B virus core antigen protein with respect to assembly        into virus-like particles, preferably into the conventional        shape of hepatitis B virus core antigen virus-like particles,        i.e., being composed of 180 or 240 subunits and having an        icosahedral structure.

In a particularly preferred embodiment, the HBcAg protein or the portionthereof has a truncation at the carboxy-terminus at an amino acidposition up to and including the position 140 of SEQ ID NO: 1 or acorresponding amino acid position. For example, the carboxy-terminaltruncation may be at and includes position 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182, or 183 of SEQ ID NO: 1 or acorresponding amino acid. A carboxy-terminal truncation at and includingposition 140 of SEQ ID NO: 1 means that the amino acids from position140 to the carboxy-terminus are missing in this particular HBcAg variantor portion, i.e., that this HBcAg protein variant or portion may consistof amino acids 1 to 139 of SEQ ID NO: 1. “A corresponding amino acidposition” in this context means that if the protein of the presentinvention comprises a HBcAg protein other than the HBcAg protein setforth in SEQ ID NO: 1, for example, a naturally occurring variant thathas an insertion, addition, and/or deletion, the amino acid at position140 of SEQ ID NO: 1 may not correspond to the amino acid at position 140of said naturally occurring HBcAg variant but to a position with a loweror higher number. The corresponding amino acids can be determined asdescribed above, for example, by sequence alignment.

In a particular preferred embodiment of the protein of the presentinvention, the hepatitis B virus core antigen protein has a truncationat the carboxy-terminus such that it is not able to bind to nucleicacids but retains the ability to assemble into virus-like particles. Themajor RNA recognizing activity was attributed to a region within theHBcAg protein corresponding to amino acids 150 to 157 of SEQ ID NO: 1.The major DNA-recognizing activity was attributed to a region within theHBcAg protein corresponding to amino acids 157 to 177 of SEQ ID NO: 1.Thus, for abolishing the nucleic acid binding ability of the HBcAgprotein, these sequences responsible for nucleic acid binding arepreferably deleted.

A particularly preferred truncation variant of the HBcAg protein used inthe present invention is a truncation at and including the amino acidposition 151 of SEQ ID NO: 1 or a corresponding amino acid position,i.e., a HBcAg protein variant which carboxy-terminal amino acidcorresponds to the amino acid at amino acid position 150 of SEQ IDNO: 1. In a preferred embodiment, said truncation variant of the HBcAgprotein further comprises a carboxy-terminal His-tag, preferably linkedto the HBcAg truncation variant via a glycine linker such as a GGSlinker. Preferably, said truncation variant of the HBcAg proteincomprises, essentially consists of, or consists of the amino acidsequence set forth in SEQ ID NO: 79. Preferably, said truncation variantof the HBcAg protein is encoded by a nucleic acid set forth in SEQ IDNO: 80.

In preferred embodiments of the protein of the present invention, all orpart of the amino acid sequence corresponding to the MIR of thehepatitis B virus core antigen protein is deleted. For example, all orpart of the amino acids 74 to 89 of SEQ ID NO: 1 may be deleted.

In a preferred embodiment of the protein of the present invention, theamino acid sequence comprising an epitope

-   -   (i) is attached to the amino-terminal amino acid of the        hepatitis B virus core antigen protein, is inserted into the 30        amino-terminal amino acid residues, preferably the 20        amino-terminal amino acid residues, preferably the 10        amino-terminal amino acid residues, preferably the 5        amino-terminal amino acid residues of the hepatitis B virus core        antigen protein, or replaces one or more, for example, 1, 2, 3,        4, 5, 6, 7, 8, 9, or 10 amino acids, of the 30 amino-terminal        amino acid residues, preferably of the 20 amino-terminal amino        acid residues, preferably of the 10 amino-terminal amino acid        residues of the hepatitis B virus core antigen protein, and/or    -   (ii) is attached to the carboxy-terminal amino acid of the        hepatitis B virus core antigen protein or is inserted into the        30 carboxy-terminal amino acid residues, preferably the 20        carboxy-terminal amino acid residues, preferably the 10        carboxy-terminal amino acid residues of the hepatitis B virus        core antigen protein, or replaces one or more, for example, 1,        2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, of the 30        carboxy-terminal amino acid residues, preferably of the 20        carboxy-terminal amino acid residues, preferably of the 10        carboxy-terminal amino acid residues of the hepatitis B virus        core antigen protein, and/or    -   (iii) is inserted into the amino acid sequence corresponding to        the MIR of the hepatitis B virus core antigen protein or        replaces one or more amino acids, for example, 1, 2, 3, 4, 5, 6,        7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, within the        amino acid sequence corresponding to the MIR of the hepatitis B        virus core antigen protein, and/or    -   (iv) is attached to one or more amino acids located within the        amino acid sequence corresponding to the MIR of the hepatitis B        virus core antigen protein, for example, to one or more of the        amino acids 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,        87, 88, or 89 of SEQ ID NO: 1 or a corresponding amino acid.

If the amino acid sequence comprising an epitope is attached to theamino-terminal amino acid of the hepatitis B virus core antigen proteinor inserted between amino-terminal residues it is preferred that theamino acid sequence comprising an epitope has a maximal length of 50amino acid residues, preferably 40 amino acid residues, preferably 30amino acid residues. It is also preferred that no more than 9,preferably no more than 5, preferably no more than 4, preferably no morethan 3 amino acids are deleted from the amino-terminus of the hepatitisB virus core antigen protein.

It is particularly preferred that the amino acid sequence comprising anepitope is inserted into the MIR or replaces one or more amino acids ofthe MIR. Preferably, the amino acid sequence comprising an epitope

-   -   (i) is inserted into the hepatitis B virus core antigen protein        between the amino acids at positions 77 and 78 of the amino acid        sequence set forth in SEQ ID NO: 1 of the sequence listing or at        a corresponding position, or    -   (ii) replaces the amino acids at positions 74-81, 76-81, 76-79,        or 79-80 of the amino acid sequence set forth in SEQ ID NO: 1 of        the sequence listing or at corresponding positions.

It is particularly preferred that the amino acid sequence comprising theepitope is inserted into or attached to the hepatitis B virus coreantigen protein such that the chimeric hepatitis B virus core antigenprotein, i.e., the hepatitis B virus core antigen protein comprising anepitope derived from a tumor-associated antigen as described above, iscapable of assembling into virus-like particles, preferably intoconventional hepatitis B virus core antigen protein virus-likeparticles. This feature of the protein can be easily determined by theskilled person, for example, by using transmission electron microscopyor asymmetric flow-field-flow fractionation combined with dynamic lightscattering, for example, as described in the Examples section of thepresent invention.

It is also preferred that the structure, in particular the length, ofthe amino acid sequence comprising the epitope is chosen such that thechimeric hepatitis B virus core antigen protein, i.e., the hepatitis Bvirus core antigen protein comprising an epitope derived from atumor-associated antigen as described above, is capable of assemblinginto virus-like particles. Thus, the amino acid sequence comprising anepitope is preferably not more than 300, preferably not more than 250,preferably not more than 200, preferably not more than 150, and morepreferably not more than 100 amino acids in length. It is preferred thatthe amino acid sequence comprising an epitope is up to 100 amino acidresidues in length, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids. It is particularlypreferred that the length of the amino acid sequence comprising anepitope is between 20 and 60 amino acids, preferably between 25 and 55amino acids, for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, or 55 amino acids.

In some embodiments of the protein of the present invention, the proteinmay in addition to the first amino acid sequence comprising an epitopecomprise one or more further amino acid sequences comprising an epitopeinserted into or attached to the hepatitis B virus core antigen protein,wherein one or more of the epitopes of the one or more further aminoacid sequences comprising an epitope are identical to or different toeach other and one or more of the epitopes of the one or more furtheramino acid sequences comprising an epitope are identical to or differentfrom the epitope of the first amino acid sequence comprising an epitope.These one or more further amino acid sequences comprising an epitope maybe inserted into or attached to the hepatitis B virus core antigenprotein as described above for the first amino acid sequence comprisingan epitope. The disclosure on the length of the first amino acidsequence comprising an epitope as well as on the epitope etc. alsoapplies to the one or more further amino acid sequences comprising anepitope. It is particularly preferred, that a chimeric hepatitis B viruscore antigen protein comprising more than one amino acid sequencescomprising an epitope is capable of assembling into virus-likeparticles.

In some embodiments of the protein of the present invention, the firstamino acid sequence comprising an epitope and/or one or more of thefurther amino acid sequences comprising an epitope comprise(s) more thanone epitope, wherein the epitopes are identical or different. Forexample, one epitope within an amino acid sequence comprising more thanone epitope may be derived from one tumor-associated antigen and theother epitope may be derived from another tumor-associated antigen. Thisis particularly advantageous if a certain type of tumor can berecognized by a combination of tumor-associated antigens. The epitopeswithin one amino acid sequence comprising more than one epitope may alsobe derived from the same tumor-associated antigen. In this case, theepitopes may be, for example, derived from different extracellular loopsor portions of the tumor-associated antigen or from the sameextracellular loop or portion.

The protein of the present invention may comprise one or more linkersequences between the individual elements making up the proteins, e.g.,the HBcAg protein derived parts and the amino acid sequence(s)comprising an epitope. Preferably, the amino acid sequence comprising anepitope further comprises a linker sequence up-stream and/or down-streamof the epitope sequence. For example, if the amino acid sequencecomprising an epitope is inserted into or replaces all or part of theMIR of the hepatitis B virus core antigen protein, in particular if itreplaces all of the MIR or a major part thereof, it is particularlypreferred that the epitope is flanked by linker sequences on each sideof the epitope.

The linker preferably consists of maximally 50 amino acids, preferablyof maximally 40 amino acids, more preferably of maximally 30 aminoacids, even more preferably of maximally 20 amino acids, and mostpreferably of maximally 10 amino acids. It is particularly preferredthat the linker consists of between 2 to 25 amino acids, preferablybetween 2 to 20 amino acids, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 amino acids, most preferably 9 aminoacids. The linker preferably comprises, preferably essentially consistsof, preferably consists of small amino acids such as glycine, alanine,or serine. Preferably, the linker is chosen such that the linked aminoacid sequences, for example, the epitope sequence, is able to assume atleast partially its native structure under native conditions. Apreferred linker essentially consists of glycine and serine residues,e.g., having a length of between 5 and 15 amino acids, for example,preferred linkers are Gly_(m)Ser_(n)Gly_(p), wherein m, n, and p areintegers independently selected from 1 to 10, wherein m+n+p is between 5and 15. A particularly preferred linker is G₄SG₄ (SEQ ID NO: 24). Inembodiments wherein the epitope sequence is flanked by linker sequences,the linker sequences may be considered to be comprised by the HBcAgportion of the protein of the present invention, by the amino acidsequence comprising an epitope portion of the protein of the presentinvention, or one of the two linker sequences may be consideredcomprised by the HBcAg portion of the protein of the present inventionand the other linker sequence by the amino acid sequence comprising anepitope portion of the protein of the present invention.

In some embodiments, the protein of the present invention may compriseone or more epitope-, peptide-, or protein-tags, for example, forfacilitating purification of the protein of the present invention. Suchepitope-, peptide-, or protein-tags include, but are not limited to,hemagglutinin- (HA-), FLAG-, myc-tag, poly-His-tag,glutathione-S-transferase- (GST-), maltose-binding-protein- (MBP-),NusA-, and thioredoxin-tag, or fluorescent protein-tags such as(enhanced) green fluorescent protein ((E)GFP), (enhanced) yellowfluorescent protein ((E)YFP), red fluorescent protein (RFP) derived fromDiscosoma species (DsRed) or monomeric (mRFP), cyan fluorescence protein(CFP), and the like. In a preferred embodiment, the epitope-, peptide-,or protein-tags can be cleaved off the protein of the present invention,for example, using a protease such as thrombin, Factor Xa, PreScission,TEV protease, and the like. The recognition sites for such proteases arewell known to the person skilled in the art. Preferably, a smallepitope- or peptide-tag is used such as a His-tag, HA-tag, or FLAG-tag,and preferably the tag can be removed. In a preferred embodiment, theprotein of the present invention comprises a His-tag, preferably aHis₆-tag, preferably at the carboxy-terminus. Preferably, the epitope-,peptide-, or protein-tag is connected to one or more of the otherelements of the protein of the present invention via a linker, whereinthe linker may be as described above. A preferred linker in this contextis the amino acid sequence GGS.

In preferred embodiments of the chimeric HBcAg proteins of the presentinvention, the HBcAg backbone sequence is selected from the groupconsisting of the amino acid sequences set forth in SEQ ID NOs: 25 to30, i.e., HBcAg backbones A, B, C, D, E, and F (FIG. 7). Preferrednucleic acid sequences encoding for the HBcAg backbones are the nucleicacid sequences set forth in SEQ ID NOs: 31 to 36, wherein SEQ ID NO: 31codes for SEQ ID NO: 25, SEQ ID NO: 32 codes for SEQ ID NO: 26, SEQ IDNO: 33 codes for SEQ ID NO: 27, SEQ ID NO: 34 codes for SEQ ID NO: 28,SEQ ID NO: 35 codes for SEQ ID NO: 29, and SEQ ID NO: 36 codes for SEQID NO: 30. The term “HBcAg backbone” relates to the portion of theprotein of the invention that is not the amino acid sequence comprisingan epitope. It is particularly preferred that these backbones arecombined with amino acid sequences comprising one or more of the epitopesequences set forth in SEQ ID NOs: 9 to 23 and 45 to 78 of the sequencelisting. For example, HBcAg backbone A (SEQ ID NO: 25) may be combinedwith an amino acid sequence comprising SEQ ID NO: 9, SEQ ID NO: 10, SEQID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 45, SEQ IDNO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55,SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO:60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ IDNO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74,SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, or SEQ ID NO: 78, HBcAgbackbone B (SEQ ID NO: 26) may be combined with an amino acid sequencecomprising SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52,SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO:57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ IDNO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71,SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO:76, SEQ ID NO: 77, or SEQ ID NO: 78, HBcAg backbone C (SEQ ID NO: 27)may be combined with an amino acid sequence comprising SEQ ID NO: 9, SEQID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54,SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO:59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ IDNO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73,SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, or SEQ IDNO: 78, HBcAg backbone D (SEQ ID NO: 28) may be combined with an aminoacid sequence comprising SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 45, SEQ ID NO: 46, SEQ IDNO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56,SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO:61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ IDNO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75,SEQ ID NO: 76, SEQ ID NO: 77, or SEQ ID NO: 78, HBcAg backbone E (SEQ IDNO: 29) may be combined with an amino acid sequence comprising SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ IDNO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58,SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ IDNO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77,or SEQ ID NO: 78, or HBcAg backbone F (SEQ ID NO: 30) may be combinedwith an amino acid sequence comprising SEQ ID NO: 9, SEQ ID NO: 10, SEQID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 45, SEQ IDNO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55,SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO:60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ IDNO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74,SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, or SEQ ID NO: 78.Preferably, the amino acid sequence comprising an epitope is insertedinto the HBcAg backbone between the linker sequence GGGGSGGGG (SEQ IDNO: 24) and the HBcAg portion, i.e., immediately upstream or downstreamof this linker sequence. Preferably, the amino acid sequence comprisingan epitope also comprises a linker sequence, wherein if the amino acidsequence comprising an epitope is inserted upstream of the linkersequence of the HBcAg backbone the linker sequence of the amino acidsequence comprising an epitope is located upstream of the epitopesequence, and if the amino acid sequence comprising an epitope isinserted downstream of the linker sequence of the HBcAg backbone thelinker sequence of the amino acid sequence comprising an epitope islocated downstream of the epitope sequence. Thus, in preferredembodiments, the epitope is flanked by linker sequences within thechimeric HBcAg proteins of the invention. It is to be understood that,for example, the His-tag located at the C-terminus of the HBcAgbackbones may be replaced by any other epitope-, peptide-, orprotein-tag as described above, and that the linker sequences may alsovary as described above.

In a particularly preferred embodiment of the protein of the presentinvention, the epitope is the CLDN18.2₃₂₋₄₁ peptide having the sequenceTQDLYNNPVT (SEQ ID NO: 21) which is flanked on both sides by a linker,preferably having the sequence Gly_(m)Ser_(n)Gly_(p), wherein m, n, andp are integers independently selected from 1 to 10, wherein m+n+p isbetween 5 and 15, more preferably having the sequence G₄SG₄ (SEQ ID NO:24). The epitope flanked on both sides by a linker (amino acid sequencecomprising an epitope) is inserted into an HBcAg backbone sequence,wherein the portion of the HBcAg backbone sequence flanking theN-terminal side of the amino acid sequence comprising an epitopepreferably comprises the amino acid sequence from positions 2 to 73, 2to 75 or 2 to 78 of SEQ ID NO: 1, preferably the amino acid sequencefrom positions 1 to 73, 1 to 75 or 1 to 78 of SEQ ID NO: 1 and theportion of the HBcAg backbone sequence flanking the C-terminal side ofthe amino acid sequence comprising an epitope preferably comprises theamino acid sequence from positions 80 to 150, 81 to 150 or 82 to 150 ofSEQ ID NO: 1. Preferably, the portion of the HBcAg backbone sequenceflanking the N-terminal side of the amino acid sequence comprising anepitope comprises the amino acid sequence from positions 2 to 78 of SEQID NO: 1, preferably the amino acid sequence from positions 1 to 78 ofSEQ ID NO: 1 and the portion of the HBcAg backbone sequence flanking theC-terminal side of the amino acid sequence comprising an epitopecomprises the amino acid sequence from positions 81 to 150 of SEQ IDNO: 1. In one embodiment, the portion of the HBcAg backbone sequenceflanking the N-terminal side of the amino acid sequence comprising anepitope may comprise additional sequences on its N-terminus and/or theportion of the HBcAg backbone sequence flanking the C-terminal side ofthe amino acid sequence comprising an epitope may comprise additionalsequences on its C-terminus.

Particularly preferred examples of the protein of the present inventionare proteins which comprise, preferably essentially consist of,preferably consist of an amino acid sequence selected from the groupconsisting of

-   -   (i) the amino acid sequences set forth in SEQ ID NOs: 37 to 40        or    -   (ii) an amino acid sequence that is at least 60%, 65%, 70%, 80%,        81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, 99%, preferably at least 80%,        identical to the amino acid sequence under (i) preferably over        60%, more preferably over at least 70%, more preferably over at        least 80%, more preferably over at least 90%, most preferably        over at least 95% of the entire length of the amino acid        sequence under (i), and is functionally, preferably        immunologically equivalent to the amino acid sequence under (i).        For example, the amino acid sequence under (ii) is preferably        able to assemble into virus-like particles and is preferably        able to elicit an immune response, preferably a humoral immune        response, in a subject against the tumor-associated antigen from        which the epitope is derived, when administered to said subject        preferably in the form of virus-like particles and without        adjuvant. As specified above, the antibodies generated are        preferably able to recognize and bind to the tumor-associated        antigen in its native conformation preferably on the surface of        a cell, preferably a tumor cell. Preferably said immune reaction        is also elicited when the tumor-associated antigen is a        self-protein in the subject.

Preferred nucleic acid sequences coding for the preferred examples ofthe protein of the present invention are the nucleic acids set forth inSEQ ID NO: 41 to 44, wherein SEQ ID NO: 41 codes for SEQ ID NO: 37, SEQID NO: 42 codes for SEQ ID NO: 38, SEQ ID NO: 43 codes for SEQ ID NO:39, and SEQ ID NO: 44 codes for SEQ ID NO: 40.

In another aspect, the present invention provides a polynucleotidecomprising, preferably essentially consisting of, preferably consistingof a nucleic acid encoding the protein of the invention. Particularlypreferred examples of nucleic acids encoding the protein of the presentinvention are set forth in SEQ ID NOs: 41 to 44. Also variants of thenucleic acid sequences encoding the protein of the invention arecontemplated by the present invention as described above, as long as theparticular protein variant encoded by the nucleic acid variant isfunctionally, preferably immunologically equivalent to the respectiveprotein. In preferred embodiments, the polynucleotides according to thepresent invention are optimized regarding the codon usage. For example,if the protein of the present invention is to be expressed in aprokaryotic host, such as E. coli, the polynucleotide encoding theprotein of the present invention is preferably optimized for prokaryoticcodon usage.

In a further aspect, the present invention provides a vector, preferablya recombinant vector, comprising the nucleic acid of the invention.“Recombinant” means that said vector does not naturally occur, forexample, that said vector has been produced by genetic engineering. Avector in the context of the present invention may be any vector knownto the skilled person including plasmid vectors, cosmid vectors, phagevectors such as lambda phage, viral vectors such as adenoviral orbaculoviral vectors, or artificial chromosome vectors such as bacterialartificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1artificial chromosomes (PAC). Said vectors include expression as well ascloning vectors. Expression vectors comprise plasmids as well as viralvectors and generally contain a desired coding sequence and appropriateDNA sequences necessary for the expression of the operably linked codingsequence in a particular host organism (e.g., bacteria, yeast, plant,insect, or mammal) or in in vitro expression systems. Cloning vectorsare generally used to engineer and amplify a certain desired DNAfragment and may lack functional sequences needed for expression of thedesired DNA fragments. The person skilled in the art is well aware oftechniques used for the incorporation of polynucleotide sequences ofinterest into vectors (also see Sambrook et al., 1989, supra). Vectors,for example, plasmids may include an origin of replication (ori), amultiple cloning site, and regulatory sequences such as promoter(constitutive or inducible), transcription initiation site, ribosomalbinding site, transcription termination site, polyadenylation signal,and selection marker such as antibiotic resistance or auxotrophic markerbased on complementation of a mutation or deletion. In one embodiment,the polynucleotide sequence of interest is operably linked to theregulatory sequences.

In a further aspect, the present invention provides a host cellcomprising the polynucleotide of the invention or the vector of theinvention. The host cells may be prokaryotic cells such as archea orbacterial cells or eukaryotic cells such as yeast, plant, insect, ormammalian cells. In a preferred embodiment, the host cell is a bacterialcell such as an E. coli cell. The person skilled in the art is wellaware of methods for introducing said polynucleotide or said vector intosaid host cell. For example, bacterial cells can be readily transformedusing, for example, chemical transformation, e.g., the calcium chloridemethod, or electroporation. Yeast cells may be transformed, for example,using the lithium acetate transformation method or electroporation.Other eukaryotic cells can be transfected, for example, usingcommercially available liposome-based transfection kits such asLipofectamine™ (Invitrogen), commercially available lipid-basedtransfection kits such as Fugene (Roche Diagnostics), polyethyleneglycol-based transfection, calcium phosphate precipitation, gene gun(biolistic), electroporation, or viral infection. In a preferredembodiment of the invention, the host cell expresses the polynucleotideof the invention. The expressed protein may be soluble and/or expressedin inclusion bodies. The protein of the invention may be purified usingprotein purification methods well known to the person skilled in theart, optionally taking advantage of the above-mentioned epitope-,peptide-, or protein-tags. In one embodiment, the protein of the presentinvention is purified under denaturing conditions. The protein of theinvention may then be assembled in vitro into virus-like particles.

In another aspect, the present invention provides a virus-like particlecomprising multiple copies, for example, 10, 20, 30, 40, 50, 60, 70, 80,90, 100, or more copies of the protein of the present invention. In apreferred embodiment, the virus-like particle of the invention iscapable of eliciting an immune response, preferably a humoral immuneresponse directed against the tumor-associated antigen in associationwith the surface of a cell when administered without adjuvant to asubject, wherein the tumor-associated antigen is a self-protein in saidsubject. It is clear to the skilled person that the tumor-associatedantigen against which the humoral immune response is directed is thetumor-associated antigen from which the epitope is derived which iscomprised by the protein of the present invention being comprised in thevirus-like particle of the present invention.

The virus-like particle of the invention may be a conventional HBcAgvirus-like particle, for example, consisting of 180 or 240 HBcAg proteinsubunits having preferably an icosahedral structure or it may assume anyother virus-like particle structure, e.g., a spherical, globular, or rodshaped structure. Preferably, the virus-like particle consists of 180 or240 subunits and has preferably an icosahedral structure.

In one embodiment, the virus-like particle of the invention is composedof hepatitis B virus core antigen proteins and multiple copies of theprotein of the invention, wherein at least 50%, preferably at least 60%,preferably at least 70%, preferably at least 80%, more preferably atleast 90%, even more preferably at least 95% of the protein subunits arethe protein of the invention. For example, the virus-like particle mayconsist of 180 protein subunits, wherein 90, 100, 110, 120, 130, 140,150, 160, 170, or 180 subunits may be the protein of the invention. Forexample, the virus-like particle may consist of 240 protein subunits,wherein 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, or240 subunits may be the protein of the present invention. The hepatitisB virus core antigen protein in this context is preferably a naturallyoccurring hepatitis B virus core antigen protein or a portion thereof,preferably a carboxy-terminally truncated portion thereof as specifiedabove for the protein of the present invention. The hepatitis B viruscore antigen may also be a genetically manipulated variant of anaturally occurring hepatitis B virus core antigen protein or a portionthereof, preferably as long as the genetic manipulation does not changefunctional properties, in particular does not interfere with the abilityof the hepatitis B virus core antigen protein to assemble intovirus-like particles.

In a particularly preferred embodiment, 100% of the protein subunitsmaking up the virus-like particle of the invention are the protein ofthe present invention. Thus, in a particularly preferred embodiment, thevirus-like particle of the present invention is composed of multiplecopies of the protein of the present invention. The single proteinsmaking up the virus-like particle may comprise the same or differentepitopes derived from a single or different tumor-associated antigens.They may also comprise the same or a different HBcAg portion, or thesame or a different HBcAg backbone.

In another aspect, the present invention provides an immunogeniccomposition comprising the protein of the present invention, the nucleicacid of the present invention, the vector of the present invention, thehost cell of the present invention, or the virus-like particle of thepresent invention and a pharmaceutically acceptable diluent, carrier,and/or excipient. Preferably, the immunogenic composition is foreliciting an immune response, preferably a humoral immune responseagainst the tumor-associated antigen in association with the surface ofa cell in a subject, wherein the tumor-associated antigen is preferablya self-protein in said subject. It is particularly preferred that theimmunogenic composition of the present invention is free of adjuvants.Preferably, the immunogenic effect of the immunogenic composition of thepresent invention, for example, the generation of autoantibodies againstthe tumor-associated antigen from which the epitope is derived, can bealso observed when administered to a subject without the addition ofadjuvants. However, the addition of adjuvants as described above mayincrease and/or prolong the immunogenic effect.

In other aspects, the present invention provides the protein of thepresent invention, the nucleic acid of the present invention, the vectorof the present invention, the host cell of the present invention, thevirus-like particle of the present invention, or the immunogeniccomposition of the present invention for prophylactic and/or therapeutictreatment of tumors. In general, the tumors in the context of thepresent invention are preferably tumors associated with expression orabnormal expression of a tumor-associated antigen, wherein thetumor-associated antigen is as defined above. For example, thetumor-associated antigen is a differentiation antigen, a cancer/testisantigen, an antigen specific for trophoblastic tissues, or a germ linespecific antigen. Preferably, the protein of the present invention, thenucleic acid of the present invention, the vector of the presentinvention, the host cell of the present invention, the virus-likeparticle of the present invention, or the immunogenic composition of thepresent invention is for prophylactic and/or therapeutic treatment of adisease associated with expression or abnormal expression of atumor-associated antigen, wherein the epitope comprised by the proteinof the present invention, the nucleic acid of the present invention, thevector of the present invention, the host cell of the present invention,the virus-like particle of the present invention, or the immunogeniccomposition of the present invention is derived from saidtumor-associated antigen.

For example, if the protein of the present invention, the nucleic acidof the present invention, the vector of the present invention, the hostcell of the present invention, the virus-like particle of the presentinvention, or the immunogenic composition of the present inventioncomprises an epitope derived from CLDN6, the protein of the presentinvention, the nucleic acid of the present invention, the vector of thepresent invention, the host cell of the present invention, thevirus-like particle of the present invention, or the immunogeniccomposition of the present invention is for prophylactic and/ortherapeutic treatment of a disease associated with expression orabnormal expression of CLDN6, for example, a tumorigenic disease asdescribed above. Preferably, the tumorigenic disease is a cancerdisease, preferably selected from the group consisting of ovariancancer, in particular ovarian adenocarcinoma and ovarianteratocarcinoma, lung cancer, including small cell lung cancer (SCLC)and non-small cell lung cancer (NSCLC), in particular squamous cell lungcarcinoma and adenocarcinoma, gastric cancer, breast cancer, hepaticcancer, pancreatic cancer, skin cancer, in particular basal cellcarcinoma and squamous cell carcinoma, malignant melanoma, head and neckcancer, in particular malignant pleomorphic adenoma, sarcoma, inparticular synovial sarcoma and carcinosarcoma, bile duct cancer, cancerof the urinary bladder, in particular transitional cell carcinoma,kidney cancer, in particular renal cell carcinoma including clear cellrenal cell carcinoma and papillary renal cell carcinoma, colon cancer,testicular embryonal carcinoma, and placental choriocarcinoma, and themetastatic forms thereof. It is particularly preferred that the cancerdisease is selected from the group consisting of ovarian cancer, lungcancer, metastatic ovarian cancer and metastatic lung cancer.Preferably, the ovarian cancer is a carcinoma or an adenocarcinoma.Preferably, the lung cancer is a carcinoma or an adenocarcinoma, andpreferably is bronchiolar cancer such as a bronchiolar carcinoma orbronchiolar adenocarcinoma.

Furthermore, for example, if the protein of the present invention, thenucleic acid of the present invention, the vector of the presentinvention, the host cell of the present invention, the virus-likeparticle of the present invention, or the immunogenic composition of thepresent invention comprises an epitope derived from CLDN18.2, theprotein of the present invention, the nucleic acid of the presentinvention, the vector of the present invention, the host cell of thepresent invention, the virus-like particle of the present invention, orthe immunogenic composition of the present invention is for prophylacticand/or therapeutic treatment of a disease associated with expression orabnormal expression of CLDN18.2, for example, a tumorigenic disease asdescribed above. Preferably, the tumorigenic disease is a cancer,preferably a cancer selected from the group consisting of gastriccancer, esophageal cancer, pancreatic cancer, lung cancer such as nonsmall cell lung cancer (NSCLC), ovarian cancer, colon cancer, hepaticcancer, head-neck cancer, and cancers of the gallbladder, and metastasesthereof, in particular gastric cancer metastasis such as Krukenbergtumors, peritoneal metastasis, and lymph node metastasis.

Furthermore, for example, if the protein of the present invention, thenucleic acid of the present invention, the vector of the presentinvention, the host cell of the present invention, the virus-likeparticle of the present invention, or the immunogenic composition of thepresent invention comprises an epitope derived from PLAC1, the proteinof the present invention, the nucleic acid of the present invention, thevector of the present invention, the host cell of the present invention,the virus-like particle of the present invention, or the immunogeniccomposition of the present invention is for prophylactic and/ortherapeutic treatment of a disease associated with expression orabnormal expression of PLAC1, for example, a tumorigenic disease.Preferably, the tumorigenic disease is a cancer, preferably a cancerselected from the group consisting of breast cancer, lung cancer,gastric cancer, ovarian cancer, hepatocellular cancer, colon cancer,pancreatic cancer, esophageal cancer, head & neck cancer, kidney cancer,in particular renal cell carcinoma, prostate cancer, liver cancer,melanoma, sarcoma, myeloma, neuroblastoma, placental choriocarcinoma,cervical cancer, and thyroid cancer, and the metastatic forms thereof.

In another aspect, the present invention provides a method for elicitingan immune response, preferably a humoral immune response, against atumor-associated antigen in a subject, wherein the tumor-associatedantigen is preferably a self-protein in said subject, said methodcomprising administering to said subject the protein of the presentinvention, the nucleic acid of the present invention, the vector of thepresent invention, the host cell of the present invention, thevirus-like particle of the present invention, or the immunogeniccomposition of the present invention. Preferably, said subject isafflicted with a tumor or is at risk of developing a tumor, said tumorbeing preferably characterized by association of the tumor-associatedantigen with the surface of a tumor cell. Preferably, said tumor isassociated with the tumor-associated antigen of which the epitope isderived which is comprised by the protein of the present invention, thenucleic acid of the present invention, the vector of the presentinvention, the host cell of the present invention, the virus-likeparticle of the present invention, or the immunogenic composition of thepresent invention. In a preferred embodiment, the method comprisesadministering the virus-like particle or the immunogenic composition ofthe present invention. Preferably, eliciting a humoral immune responsecomprises the generation of antibodies, preferably autoantibodies, whichspecifically recognize/bind to the tumor-associated antigen from whichthe epitope is derived in association with the surface of a cell,preferably on the surface of a living cell, for example, a tumor cellwhich carries/expresses the tumor-associated antigen. Preferably, thegenerated antibodies recognize the tumor-associated antigen in itsnative conformation on the surface of a cell. It is particularlypreferred that the generated antibodies are capable of elicitingeffector functions against cells carrying the tumor-associated antigenfrom which the epitope is derived on their surface. For example, thegenerated antibodies may be capable of mediating ADCC and/or CDC againstsuch cells and/or they may directly induce apoptosis in or inhibitproliferation of the cells carrying the tumor-associated antigen ontheir surface. Preferably, in this aspect of the present invention, theimmune response, preferably the humoral immune response, results inreduction, preferably inhibition of tumor growth, and most preferably inregression of the tumor in the subject.

In a further aspect, the present invention provides a method forbreaking self-tolerance towards a tumor-associated antigen in a subject,said method comprising administering to said subject the protein of thepresent invention, the nucleic acid of the present invention, the vectorof the present invention, the host cell of the present invention, thevirus-like particle of the present invention, or the immunogeniccomposition of the present invention. Preferably, breaking theself-tolerance is with respect to the tumor-associated antigen fromwhich the epitope is derived which is comprised by the protein of thepresent invention, the nucleic acid of the present invention, the vectorof the present invention, the host cell of the present invention, thevirus-like particle of the present invention, or the immunogeniccomposition of the present invention.

In another aspect, the present invention provides a method for treatingand/or preventing a tumor in a subject, said method comprisingadministering to said subject the protein of the present invention, thenucleic acid of the present invention, the vector of the presentinvention, the host cell of the present invention, the virus-likeparticle of the present invention, or the immunogenic composition of thepresent invention. Preferably, said tumor is associated with expressionor abnormal expression the tumor-associated antigen from which theepitope is derived that is comprised in the protein of the presentinvention, the nucleic acid of the present invention, the vector of thepresent invention, the host cell of the present invention, thevirus-like particle of the present invention, or the immunogeniccomposition of the present invention. The particular preferred tumortypes treated and/or prevented are as described herein, in particular,as described for the exemplary tumor-associated antigens CLDN6,CLDN18.2, and PLAC1.

In preferred embodiments of the methods and uses of the presentinvention the protein of the present invention, the nucleic acid of thepresent invention, the vector of the present invention, the host cell ofthe present invention, the virus-like particle of the present invention,or the immunogenic composition of the present invention is administeredwithout adjuvant.

The present invention also provides the use of the protein of thepresent invention, the nucleic acid of the present invention, the vectorof the present invention, the host cell of the present invention, thevirus-like particle of the present invention, or the immunogeniccomposition of the present invention for the preparation of a medicamentfor prophylactic and/or therapeutic treatment of a tumor, for elicitinga humoral immune response against a tumor-associated antigen in asubject, wherein the tumor-associated antigen is a self-protein in saidsubject, or for breaking self-tolerance towards a tumor-associatedantigen in a subject. In this context, the above described embodimentsalso apply to this aspect of the present invention.

For all the above methods and uses, the protein of the presentinvention, the nucleic acid of the present invention, the vector of thepresent invention, the host cell of the present invention, thevirus-like particle of the present invention, or the immunogeniccomposition of the present invention is preferably administered to asubject in need thereof in a therapeutically effective amount. It ispreferred that the compounds and compositions described herein areadministered orally, buccally, sublingually, intranasally, via pulmonaryroutes such as by inhalation, via rectal routes, or parenterally, forexample, intracavernosally, intravenously, intra-arterially,intraperitoneally, intrathecally, intraventricularly, intra-urethrallyintrasternally, intracranially, intramuscularly, intradermally,intranodally, or subcutaneously. Administration may be by infusion orneedleless injection techniques. Preferably, the compounds orcompositions described herein are administered parenterally. Acomposition suitable for parenteral administration is best used in theform of a sterile aqueous solution which may contain other substances,for example, enough salts or glucose to make the solution isotonic withblood. The aqueous solutions should be suitably buffered (preferably toa pH of from 3 to 9), if necessary.

All methods and uses of the present invention preferably aim at theprophylactic and/or therapeutic treatment of a tumorigenic disease asdescribed herein above. In some embodiments, said methods and uses ofthe present invention may be combined with conventional tumor therapy,such as surgery, radiation therapy, chemotherapy, and/or passiveimmunization with monoclonal antibodies. For example, the compounds,compositions, methods, or uses of the present invention may be appliedafter surgical removal of the primary tumor in order to target tumorcells that have not been excised and/or to prevent formation ofmetastasis. The compounds and compositions described herein may also bepart of a composition used for chemotherapy, for example, they may becomprised by a conventional chemotherapeutic composition.

The present inventors have achieved to provide means for activeimmunization of a subject against tumorigenic diseases which areassociated with the expression or abnormal expression oftumor-associated antigens, which are self-proteins in said subject, andthus, provide means and methods for preventing and/or treating suchtumorigenic diseases.

The present invention is described in detail by the figures and examplesbelow, which are used only for illustration purposes and are not meantto be limiting. Owing to the description and the examples, furtherembodiments which are likewise included in the invention are accessibleto the skilled worker.

EXAMPLES Generation of the HBcAg-VLP Based Vaccines

For recombinant generation of chimeric HBcAg fusion proteins variousbacterial expression vectors (HBcAg backbones) have been generated,which differ regarding the epitope insertion sites within the HBcAgsequence. In any of the generated HBcAg backbones epitopes may beinserted in specific regions of the HBcAg MIR. With the exception of theHBcAg backbones HBcAg Del 79-80 linker and HBcAg Del 79-80 epitopes mayadditionally be attached to the amino-terminus or inserted into theamino-terminal part of the HBcAg protein, for example, between the SalIand SpeI restriction sites (FIG. 1). The wild type sequence of theN-terminal part of the HBc gene subtype awy is MDIDPYK. The insertion ofthe restriction sites SalI and SpeI leads to the sequence M VDAATSDIDPYK, wherein the alanines are needed for the separation of therestriction sites. The insertion of an epitope (xxx) between therestriction sites SalI and SpeI would result in the sequence M VE xxx SSDIDPYK. The DNA sequences of the fusion proteins have beensequence-optimized for the expression in E. coli. Appropriaterecognition sequences for restriction enzymes have been integrated inthe expression cassettes for the fusion protein, which allow replacingor integrating the various regions of the cassette or the epitope to beinserted without major effort (cf. FIGS. 1 and 7).

CLDN6, CLDN18.2, and PLAC1 epitopes, respectively, either have beendirectly inserted into the HBcAg MIR (HBcAg backbone HBcAg Del 79-80),or have been flanked amino- and carboxy-terminally by a glycine/serine(G₄SG₄) linker for increasing epitope flexibility during proteinfolding. Furthermore, a sequence coding for a histidine tag (His-tag)has been integrated at the 3′-end of the expression cassette allowing asubsequent purification of the fusion protein under GMP conditions usingaffinity chromatography (cf. FIG. 1). The utilized HBcAg sequences are3′-truncated variants which code for a C-terminally truncated HBcAgprotein (aa 1-150). This variant is capable of assembling into VLPs andis not able to bind nucleic acids in contrast to the wild-type HBcAg (aa1-183) preventing a potential contamination of the vaccine withbacterial nucleic acids.

After expression of the chimeric HBcAg fusion constructs in E. coli thefusion proteins have been purified in their dimeric form underdenaturing conditions using immobilized metal ion affinitychromatography. Subsequently the in vitro assembly of the fusionproteins into VLPs has been performed using dialysis against high saltbuffer and a final sucrose density gradient ultracentrifugation step forfurther purification and concentration of assembled VLPs. The successfulreassembly and quality of the VLPs has been verified using nativeprotein agarose gel electrophoresis and negative contrast transmissionelectron microscopy (cf. FIG. 2). It has been verified that specificepitopes of the tumor antigens CLDN6, CLDN18.2, and PLAC1 may beinserted into HBcAg without interfering with the assembly competence ofHBcAg into VLPs. The fusion proteins which have been purified usingdenaturing methods have been in vitro assembled into highly pure VLPswhich did not differ in their electron microscopic appearance fromwild-type HBcAg VLPs. The results of further immunization experimentsare subsequently exemplarily shown for the following chimeric HBcAg VLPs(cf. FIG. 8):

HBcAg Del 79-80 linker CLDN18.2-EC1 short (SEQ ID NOs: 37 and 41):Fusion protein consisting of an amino- and carboxy-terminal HBcAg domain(expression vector HBcAg Del 79-80), an inserted and glycine linkerflanked CLDN18.2 epitope (TQDLYNNPVT; SEQ ID NO: 21) of theextracellular domain 1 (EC1) and a C-terminal His-tag.

HBcAg Del 79-80 CLDN18.2-EC1 short (SEQ ID NOs: 38 and 42): Fusionprotein consisting of an amino-terminal and carboxy-terminal HBcAgdomain (expression vector HBcAg Del 79-80), an inserted CLDN18.2 epitope(TQDLYNNPVT; SEQ ID NO: 21) of the extracellular domain 1 (EC1), and aC-terminal His-tag.

HBcAg Del 79-80 linker PLAC1 3^(rd) Loop A (SEQ ID NOs: 39 and 43):Fusion protein consisting of an amino-terminal and carboxy-terminalHBcAg domain (expression vector HBcAg Del 79-80), an inserted andglycine linker flanked PLAC1 epitope (VFSEEEHTQVP; SEQ ID NO: 22) of thepredicted third PLAC1 protein loop and a C-terminal His-tag.

HBcAg Del 79-80 PLAC1 3^(rd) Loop B (SEQ ID NOs: 40 and 44): Fusionprotein consisting of an amino-terminal and carboxy-terminal HBcAgdomain (expression vector HBcAg Del 79-80), an inserted PLAC1 epitope(VFSEEEHTQV; SEQ ID NO: 23) of the predicted third PLAC1 protein loopand a C-terminal His-tag.

Verification of the Chimeric HBcAg-VLP Based Vaccines

For verification of the general various species spanning immunogenicityand antigenicity of the purified chimeric HBcAg VLPs they have beenapplied to NZW rabbits and Balb/c mice (only CLDN18.2 epitope carryingVLPs), respectively. The immunization studies have been carried out withand without addition of adjuvants.

The sequence of the CLDN18.2 epitope (SEQ ID NO: 21) is identical to therespective region of the endogenously expressed protein in rabbit andmouse. The induction of an antibody response against CLDN18.2 thus wouldindicate the breaking of self-tolerance in the respective organism.Indirect immunofluorescence assays (IF) as well as flow cytometricanalyses (FACS) have been used as read-out for characterization andvalidation of the induced humoral immune responses.

CHO cells transiently transfected with CLDN18.2 and PLAC1, respectively,have been used for indirect IFs. The cells have been fixed on slides,permeabilized in some cases (only for CLDN18.2), and incubatedsubsequently with the respective polyclonal antisera. The immunologicaldetection of bound antibodies has been carried out using fluorescencelabeled secondary antibodies. Non-transfected CHO cells or transfectedCHO cells only incubated with secondary antibodies have been used asnegative controls. Furthermore, the CLDN18.2 directed polyclonalantisera have been tested regarding their specificity using CHO cellswhich have been transfected with CLDN18.1, a splice variant of CLDN18which differs from CLDN18.2 in the amino-terminal amino acids 1-69 (cf.FIG. 3).

It is shown that the generated polyclonal antisera were able torecognize the respective targeted surface antigens in their nativeconformation and that these antibodies can bind to said surface antigens(exemplarily shown in FIG. 3 for CLDN18.2 and PLAC1). It was irrelevantwhether the immunogens have been applied with or without adjuvants.Furthermore, it is demonstrated for the CLDN18.2 epitope carrying HBcAgVLPs that they are capable of breaking self-tolerance in two differentspecies. Furthermore, the antisera which have been generated byimmunization with the CLDN18.2 epitope exhibited an isoform specificimmune reactivity against CLDN18.2 transfected cells.

FACS analyses have been performed as a further approach for verifyingthe immunoreactivity of the generated polyclonal antisera (exemplarilyshown for CLDN18.2; cf. FIG. 4). Contrary to the indirect IF, however,the cells neither have been fixed nor permeabilized in theseexperiments. The detection of the antigens is thus carried out in thenative conformation on live cells. Furthermore, cells endogenouslyexpressing the targeted tumor-associated antigen have been used, wherebyit has been verified whether the generated antisera are able to detectphysiological densities of tumor-associated antigen epitopes (cf. FIG.4).

It is shown that the generated CLDN18.2 directed polyclonal antiserawere able to detect endogenously expressed antigens and to specificallybind the protein in its native conformation on live cells. It has beenverified that the existing B cell tolerance against the self protein canbe broken by active vaccination, wherein the addition of adjuvants tothe immunogen was irrelevant.

Besides the recognition of the native conformation of the targeted cellsurface antigen, the induction of antibodies with therapeuticallyeffective effector functions is of utmost importance for a successfulactive immunization strategy. Antibody effector functions are on the onehand cytotoxic effects, e.g., complement dependent cytotoxicity (CDC) orantibody dependent cellular cytotoxicity (ADCC) as well asantibody-mediated anti-proliferative effects. Luciferase based CDC andADCC assays, respectively, have been used for the analysis of cytotoxiceffector functions of the CLDN18.2 directed polyclonal antisera. It isshown that the antisera generated in two different species exhibited CDCas well as ADCC effector functions which were specifically directedagainst the CLDN18.2 isoform. Polyclonal antisera which have beeninduced by immunization with HBcAg wild-type VLPs (without insertedantigen epitopes) or Keyhole Limpet Hemocyanin (KLH)-conjugated CLDN18.2peptides (which have been identical to the epitopes inserted in thechimeric HBcAg VLPs) in combination with adjuvants did not exhibit anycytotoxic effector functions (cf. FIG. 5).

Previous studies have been shown that the expression of PLAC1 issupporting proliferation. Thus, the PLAC1 directed polyclonal antiserawhich have been generated by active immunization have been analyzedregarding proliferation inhibiting effector functions. To this end,endogenously PLAC1 expressing cells (MCF-7) as well as PLAC1 negativecells (MelHO) have been incubated with polyclonal PLAC1 directedantisera for 72 hours and subsequently a 5-bromo-2′-deoxyuridine (BrdU)based proliferation assay has been performed. A monoclonal antibodydirected against the oncogene myc and a polyclonal antiserum againstCLDN18.2 which has been generated by active vaccination with chimericHBcAg VLPs, have been used as controls. It is shown that only thePLAC1-directed polyclonal antisera mediated dosage-dependentPLAC1-specific anti-proliferative effects. Thus, besides cytotoxiceffector functions also anti-proliferative antibody effector functionsmay be generated by active immunization using chimeric HBcAg VLPs (cf.FIG. 6).

Immunization with HBcAg CLDN18.2-EC1 Short-VLPs

The potency of chimeric VLPs to induce antibody responses against theinserted CLDN18.2 epitope was analyzed by immunization of mice andrabbits. Importantly, the selected epitope and the tissue distributionof the orthologous proteins with strict restriction to short livedgastric cells are conserved in all three species.

An almost maximal anti-target humoral immune response as measured byflow cytometry was observed after only two immunizations, irrespectiveof the immunization route or applied adjuvants. Analysis of thetarget-specific antibody response revealed that anti-target antibodyreactivity decreased over time and was back to background levelsapproximately two months after the third vaccination (vaccination at d0,d10 and d28). However, when a booster was given at d102, theauto-antibody reactivity to the target increased rapidly, suggestingexistence of immune memory for auto-antibody production. Moreover, thisdata indicates that induction of target-specific auto-antibodies bybooster immunizations are feasible and that the anti-HBcAg directedimmunity had not taken over.

In a further experiment, partly related to the question of antibodyresponse drift, it could be demonstrated that a pre-existing immuneresponse against the HBcAg-carrier molecule does not abrogate theability of HBcAg Del 79-80 linker CLDN18.2-EC1 short-VLPs to inducetarget-specific auto-antibody responses. Thus, potentially existingcarrier induced epitopic suppression (CIES) can be overcome, indicatingthat the immunogenicity of the CLDN18.2 epitope displayed at highdensity on the surface of the HBcAg carrier (240 CLDN18.2 epitopes perVLP molecule) is in this context comparable to the backbone itself.

BALB/c mice were immunized with HBcAg Del 79-80 CLDN18.2-EC1 short-,HBcAg Del 79-80 linker CLDN18.2-EC1 short-VLPs or with KLH-conjugatedlinear CLDN18.2₃₂₋₄₁ peptide as control and antibody reactivity againstthe linear CLDN18.2₃₂₋₄₁ peptide or the HBcAg backbone was determined bymeasuring ELISA endpoint titer. Different immunization protocols wereapplied, varying the adjuvant, administration route and immunogen amounteach to groups of 3-5 mice.

It was observed that sera from mice immunized with HBcAg Del 79-80linker CLDN18.2-EC1 short-VLPs displayed a higher specific reactivityagainst the linear BSA-conjugated CLDN18.2₃₂₋₄₁ peptide as compared tomice in other groups. Interestingly, mice immunized s.c. with HBcAg Del79-80 linker CLDN18.2-EC1 short-VLPs without the addition of adjuvantrevealed the highest mean endpoint titer against the peptide. All VLPsinduced antibodies against the HBcAg backbone with similar endpointtiters. Heat denaturation of chimeric VLPs abrogated their capability toinduce peptide-binding antibodies without compromising development ofantibodies against the backbone.

Of the mice vaccinated with HBcAg Del 79-80 linker CLDN18.2-EC1short-VLPs ˜90% were shown to recognize the linear CLDN18.2 epitope inELISA. One third of these were able to bind to the native CLDN18.2molecule on transfectants as analyzed by FACS. In rabbits all sera ofanimals vaccinated with HBcAg Del 79-80 linker CLDN18.2-EC1 short-VLPswere able to recognize the linear epitope as well as the native protein.When using CLDN18.1 transfectants as control, no cross reactivity withthis variant was observed.

Compared to HBcAg Del 79-80 CLDN18.2-EC1 short-VLPs, HBcAg Del 79-80linker CLDN18.2-EC1 short-VLPs were clearly superior in elicitingauto-antibodies, which recognize the native protein in physiologicaldensities on the surface of endogenously expressing tumor cells.

Prophylactic Vaccination with HBcAg Del 79-80 Linker CLDN18.2-EC1Short-VLPs Confers Partial Protection in an Immunocompetent SyngeneicMouse Tumor Model

To evaluate prophylactic in vivo efficacy of HBcAg Del 79-80 linkerCLDN18.2-EC1 short-VLPs, a syngeneic tumor model in immunocompetentBALB/c mice in which pulmonary metastasis formation was induced by i.v.application of CT26 colonic cancer cells stably transduced with murineCLDN18.2 was used.

BALB/c mice were vaccinated three times (day 1, day 14, day 28) with 50μg C-terminally truncated (amino acids 1-150) HBcAg (HBcAgΔ)-VLPs, HBcAgDel 79-80 linker CLDN18.2-EC1 short-VLPs, or PBS as control, allformulated in AbISCO-100 (Isconova). Two weeks after the lastimmunization 1×10⁵ syngeneic CT26 colon cancer cells stably expressingmurine CLDN18.2 were administered into the tail vein. Thirteen dayslater mice were sacrificed and lungs weighed and subjected tomicroscopic analysis to assess load of pulmonary metastases. Statisticalanalysis of lung weights was performed by ANOVA followed by Tukey test.For histopathological assessment, three μm thick sections of formalinfixed and paraffin embedded lungs were deparaffinized and rehydrated,followed by heat induced epitope retrieval in citrate buffer at pH 6.After quenching of endogenous peroxidases by H₂O₂, unspecific antibodybinding sites were blocked with 10% goat serum, followed by overnightincubation with polyclonal rabbit anti-CLDN18 (Mid) (Invitrogen) at 4°C. For detection of binding, a HRP-conjugated secondary antibody(BrightVision Poly-HRP-Anti-rabbit, Immunologic) and the Vector NovaRED™kit (Vector Laboratories) were used. After hematoxylin counterstaining,dehydration and mounting, sections were documented using a MIRAX SCAN(Zeiss). Ratios of tumor and normal tissue areas were determined usingImageJ Software v.1.44. Statistical differences between groups wereassessed by ANOVA followed by Dunn's test.

Macroscopic analysis of lungs derived from mice vaccinated with HBcAgDel 79-80 linker CLDN18.2-EC1 short-VLPs revealed a smaller number ofmetastatic nodules as compared to HBcAgΔ-VLPs or PBS control groups(FIG. 9A) and significantly lower lung weights close to those of micenot challenged with tumor cells (FIG. 9B). Moreover, the percentage ofcancerous tissue area per whole lung section as calculated aftervisualizing CT26-CLDN18.2 pulmonary metastases by IHC-staining forCLDN18.2 was significantly (p<0.05) smaller as compared to micevaccinated with HBcAgΔ-VLPs or PBS control groups (FIG. 9C, 9D).

In conclusion, these data show that prophylactic vaccination with HBcAgDel 79-80 linker CLDN18.2-EC1 short-VLPs mediates protection againsthighly malignant/tumorigenic CT26-CLDN18.2 cells.

In summary, the developed chimeric HBcAg VLPs fulfill all requirementsfor the use in an active immunotherapeutically effective tumorvaccination. It has been shown that by administration of the chimericHBcAg VLPs, antibodies can be generated in a subject againsttumor-associated antigens which are self-proteins in said subject, andthat the induced antisera mediate therapeutically effective effectorfunctions.

1. A protein comprising all or a portion of the amino acid sequence of ahepatitis B virus core antigen protein and inserted therein or attachedthereto an amino acid sequence comprising an epitope, wherein theepitope is derived from an extracellular portion of a tumor-associatedantigen associated with the surface of a tumor cell.
 2. The protein ofclaim 1, which is capable of eliciting a humoral immune responsedirected against the tumor-associated antigen in association with thesurface of a cell when administered in the form of a virus-like particlewithout adjuvant to a subject, wherein the tumor-associated antigen is aself-protein in said subject.
 3. The protein of claim 1, wherein thetumor-associated antigen is a protein of the claudin family or PLAC1,wherein the protein of the claudin family is preferably selected fromthe group consisting of CLDN18.2 and CLDN6.
 4. The protein of claim 1,wherein the epitope comprises (i) an amino acid sequence which isselected from the group consisting of the amino acid sequences set forthin SEQ ID NOs: 9 to 23 and 45 to 78 of the sequence listing, (ii) anamino acid sequence that is at least 80% identical to and isimmunologically equivalent to the amino acid sequence under (i), or(iii) an amino acid sequence under (i) or (ii) which is truncated and isimmunologically equivalent to the amino acid sequence under (i).
 5. Theprotein of claim 1, wherein the hepatitis B virus core antigen proteincomprises an amino acid sequence selected from the group consisting of(i) the amino acid sequence set forth in SEQ ID NO: 1 or a portionthereof of at least 50 amino acids, or (ii) an amino acid sequence thatis at least 80% identical to the amino acid sequence or the portionthereof under (i).
 6. The protein of claim 1, wherein the amino acidsequence comprising an epitope (i) is attached to the amino-terminalamino acid of the hepatitis B virus core antigen protein, and/or (ii) isattached to the carboxy-terminal amino acid of the hepatitis B viruscore antigen protein, and/or (iii) is inserted into or replaces one ormore of the amino acids within the amino acid sequence corresponding tothe MIR of the hepatitis B virus core antigen protein, and/or (iv) isattached to one or more amino acids located within the amino acidsequence corresponding to the MIR of the hepatitis B virus core antigenprotein.
 7. The protein of claim 1, wherein the amino acid sequencecomprising an epitope (i) is inserted into the hepatitis B virus coreantigen protein between the amino acids at positions 77 and 78 of theamino acid sequence set forth in SEQ ID NO: 1 of the sequence listing orat a corresponding position, or (ii) replaces the amino acids atpositions 74-81, 76-81, 76-79, or 79-80 of the amino acid sequence setforth in SEQ ID NO: 1 of the sequence listing or at correspondingpositions.
 8. The protein of claim 1, which comprises an amino acidsequence selected from the group consisting of (i) the amino acidsequences set forth in SEQ ID NOs: 37 to 40 or (ii) an amino acidsequence that is at least 80% identical to and is immunologicallyequivalent to the amino acid sequence under (i).
 9. A nucleic acidencoding the protein of claim
 1. 10. A vector comprising the nucleicacid of claim
 9. 11. A host cell comprising the nucleic acid of claim 9.12. A virus-like particle comprising multiple copies of the protein ofclaim
 1. 13. The virus-like particle of claim 12, which is capable ofeliciting a humoral immune response directed against thetumor-associated antigen in association with the surface of a cell whenadministered without adjuvant to a subject, wherein the tumor-associatedantigen is a self-protein in said subject.
 14. An immunogeniccomposition comprising the protein of claim 1 and a pharmaceuticallyacceptable diluent, carrier, and/or excipient, wherein the immunogeniccomposition is preferably free of adjuvants.
 15. The immunogeniccomposition of claim 14 for eliciting a humoral immune response againstthe tumor-associated antigen in association with the surface of a cellin a subject, wherein the tumor-associated antigen is a self-protein insaid subject.
 16. The protein of claim 1 for prophylactic and/ortherapeutic treatment of tumors.
 17. A method for eliciting a humoralimmune response against a tumor-associated antigen in a subject, whereinthe tumor-associated antigen is a self-protein in said subject, saidmethod comprising administering to said subject the protein of claim 1,wherein said subject is afflicted with a tumor or is at risk ofdeveloping a tumor, said tumor being characterized by association of thetumor-associated antigen with the surface of a tumor cell, whereinadministering is preferably without adjuvant.
 18. A method for breakingself-tolerance towards a tumor-associated antigen in a subject or fortreating and/or preventing a tumor in a subject, said method comprisingadministering to said subject the protein of claim 1, whereinadministering is preferably without adjuvant.